EVER2 controls NF-κB and JNK/AP-1 pathways.

<p>(A–D) Cell lines were left untreated (-) or were treated with specific inhibitors of JNK (SP60025 (SP; 20 µM)), PI3K (Ly294002 (Ly; 15 µM)), IKKβ (Bay11-7082 (Bay; 5 µM)), PKCα (Gö6976 (Gö; 0.5 µM)), AKT1/2 (Akti; 2 µM), EGF receptor (AG1478 (AG; 12.5 µM) or N-acetylcysteine (NAC; 10 µM) as indicated. EV and Healthy cell lines were left unstimulated (A) or were stimulated with TNF (B) in the presence of brefeldin A for 16 hours. Whole-cell lysates were assayed for intracellular IL-6 by western blotting. The data shown are from one experiment representative of two independent experiments carried out. The IL-6 and actin bands on western blots were quantified by densitometry. Results are reported as the ratio of IL-6 to actin. In panel A, the ratio obtained for untreated EV cells was set to 1. In panel B, the ratio was set to 1 for each cell line in the absence of inhibitors. (C) miEVER2 and miCTRL cell lines were left untreated (-) or were treated with inhibitors specific as indicated. We analyzed the levels of c-jun or its phosphorylated form (phc-jun) in nuclear extracts by western blotting. The results shown are representative of two independent experiments. (D) EV and Healthy cell lines were transiently transfected with 0.3 µg/well HPV5-flLCR/luc or HPV5-minLCR/luc. Cells were assayed for luciferase activity. Results are reported as fold-increases over the level obtained with HPV5-minLCR/luc. Values obtained with the unstimulated Healthy cell line are set to 100%. The data shown are means ± SD of three independent experiments. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

EVER2 deficiency impairs TRAF protein production.

<p>(A–D) EV and Healthy cell lines were left untreated or stimulated with TNF for the time periods indicated. (A) Cytoplasmic extracts were subjected to western blotting. The results shown are representative of three independent experiments. (B) Whole-cell lysates were analyzed by western blotting after various times. (C) qRT-PCR was used to measure the induction of <i>TRAF1</i> 1.5–12 h after stimulation with TNF. Results are reported as fold-induction with respect to untreated cells. The basal level for the Healthy cell line was set to 1. A logarithmic (log) scale was used for the <i>Y</i> axis. Experiments were performed twice independently. (D) Nuclear extracts were subjected to immunoblot analysis with TRAF2 antibody for the times indicated. The results shown are representative of three independent experiments. (E–F) EV and Healthy (Heal.) cell lines were left untreated. (E) Cells were stained for TRAF2; the nucleus was stained with DAPI. Bars, 20 µm. (F) Keratinocytes were lysed in Triton X-100 lysis buffer. Soluble and insoluble fractions were subjected to immunoblot analysis with TRAF2 antibody. The TRAF2 and actin bands were quantified by densitometry for the soluble fraction. Results are reported as the ratio of TRAF2 to actin. The ratio obtained for the Healthy cell line was set to 1. (G) HEK-293T cells were transfected with plasmids encoding Flag-tagged EVER2 and Gluc2-tagged TRAF1, TRAF2 or RIPK1 proteins or Gluc2 fragment as a control (Gluc2-Ø). Crude lysates were immunoprecipitated with anti-Flag antibody (IP) and immunoblotted (IB) with the antibodies indicated. (H) Endogenous TRAF2 was immunoprecipitated (IP) with anti-TRAF2 in miEVER2 and control cells, and then subjected to immunoblotting (IB) with anti-Ubiquitin (Ub) antibody. The results shown are representative of three independent experiments. (I) HEK-293T cells were cotransfected with Gfp-tagged TRAF2, hemaglutinin (HA)-tagged Ubiquitin (Ub) with or without Flag-EVER2. After 24 h, the cells were treated with TNF for 2 h. Lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. The results shown are representative of two independent experiments.</p

Characterization of cell lines.

<p>(A) EV and Healthy cell lines were compared with fibroblasts. They were studied by phase-contrast microscopy. Bars, 50 µm. They were also stained for cytokeratin (KL1); the nucleus was stained with DAPI. Bars, 100 µm. (B–C) <i>EVER2</i> expression was determined in EVER2^−/− cell lines (EV and miEVER2) compared with control cell lines (Healthy and miCTRL). <i>EVER2</i> transcripts (B) were studied by semiquantitative RT-PCR. Healthy cells transfected with control siRNA (siCTRL) were also compared with cells transfected with siRNAs targeting various exons (5, 6, 8 and 10) of <i>EVER2</i> (siEVER2). The levels of transcripts after various numbers of cycles are shown for RT-PCR studies. <i>GAPDH</i> transcripts were used as the reference. The production of EVER2 protein (C) was evaluated by the western blotting of insoluble fractions isolated from the cells. The data shown are representative of three independent experiments. (D–E) IL-6 production was studied in EVER2^−/− (EV and miEVER2) and wild-type (Healthy or miCTRL) cells. The cells were cultured in the presence or absence of TNF. (D) After 48 h, the supernatants were collected and tested for IL-6 and IL-8 by flow cytometry. Data are means ± SD of three independent experiments. (E) After 16 h in the presence of brefeldin A, whole-cell lysates were assayed for intracellular IL-6 by western blotting. Data are from one experiment representative of three independent experiments carried out. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

EVER2 regulates the transcriptional activity of NF-κB.

<p>(A–B) Keratinocyte cell lines were transiently transfected with 0.3 µg/well NF-κB luciferase reporter. The cells were serum-starved overnight and left untreated or were stimulated with TNF (A) or a combination of PMA+ionomycin (B). The cells were harvested and assayed for luciferase activity. The data shown are expressed as fold-induction with respect to nonstimulated cells. Values obtained with wild type cells were taken as 100%. The data shown are means ± SD of three independent experiments. (C) EV or healthy cells were transiently transfected with the NF-κB/luc construct (0.3 µg/well) and various amounts of a plasmid encoding EVER2 or an empty vector as a control. Cells were assayed for luciferase activity after stimulation with TNF following serum starvation. The data shown are expressed as fold-induction with respect to nonstimulated cells. Values obtained with healthy cells transfected with empty vector were taken as 100%. The data shown are means ± SD of three independent experiments. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Function of EVER2 in NF-κB and c-jun activation.

<p>miEVER2 and miCTRL cell lines were left untreated or were stimulated with PMA+ionomycin (A) or TNF (B–J) for the times indicated. (A) Whole-cell lysates were analyzed by western blotting to explore the IKKαβ complex. The phosphorylated forms of IKKαβ are indicated as phIKKαβ. The IKKα and actin bands on western blots were quantified by densitometry. Results are reported as the ratio of IKKα to actin. The ratio obtained for the miCTRL cell line at time 0 was set to 1. (B–C) Cytoplasmic and nuclear fractions were analyzed for the classical NF-κB and AP-1 axis (B) or for the alternative NF-κB axis (C). The phosphorylated forms of p65 and c-jun are indicated as php65, and phc-jun respectively. Whole-cell lysates (Total) were analyzed for total c-jun expression. (D–H) Bands on western blots were quantified by densitometry. (D–E) The amounts of p65 and phosphorylated p65 (php65) were quantified in nuclear and cytoplasmic fractions, following stimulation with TNF. The results are reported as the ratio of nuclear and cytoplasmic concentrations (D). The ratios for basal levels were set to 1. (E) The amounts of p65 and php65 in nuclear fractions are reported. Basal levels of miCTRL were set to 1. (F–H) Subcellular extracts were assayed for p50 and the phosphorylated form of c-jun (phc-jun). For p50, the results are reported as the ratio of nuclear and cytoplasmic concentrations (F). Ratios for basal levels were set to 1. Total amounts of p50 (G) and phosphorylated c-jun (phc-jun) (H) in the nuclear fraction of cells are reported. Basal levels of miCTRL were set to 1. Data are means ± SD of three independent experiments. Statistically significant differences between the results for two cell lines are indicated by asterisks over the brackets. Asterisks over error bars indicate statistically significant differences in the results between unstimulated and TNF-stimulated cells. (I–J) Keratinocytes from the miEVER or the miCTRL cell lines were left untreated or were treated with TNF for 1 hour and stained for NF-κB (p65); the nucleus was stained with DAPI (I). Bars, 20 µm. (J) The fluorescence intensity of nuclear p65 was quantified before and after stimulation. Asterisks over error bars indicate statistically significant differences in fluorescence intensity between unstimulated and TNF-stimulated cells. Statistically significant differences between the two cell lines are indicated by asterisks over the brackets. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Image_5_Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.tif

IntroductionPlasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites.MethodsConsidering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development.Results and DiscussionThough IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.</p

Table_3_Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.xlsx

IntroductionPlasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites.MethodsConsidering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development.Results and DiscussionThough IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.</p

Image_6_Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.tif

IntroductionPlasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites.MethodsConsidering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development.Results and DiscussionThough IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.</p

Table_2_Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.xlsx

IntroductionPlasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites.MethodsConsidering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development.Results and DiscussionThough IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.</p

Image_1_Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.tif

IntroductionPlasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites.MethodsConsidering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development.Results and DiscussionThough IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.</p