- Email: [email protected]
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Abstract / Cytokine 70 (2014) 28–79
Results: While the highest CD8 T cell responses were observed after vaccination with rAd vectors with high expression of antigen, the level of antigen expression inversely correlated with innate immune responses. Upon interrogation of candidate innate genes, augmenting type I IFN signaling decreased antigen expression while abrogating type I IFN signaling increased antigen expression and promoted early CD8 T cell expansion. More specifically and upstream of type I IFN induction, deficiency in STING but not RLR signaling pathways increased antigen expression and CD8 T cell expansion. Conclusions: These data illustrate a reciprocal relationship between antigen expression and innate immune responses for CD8 T cell immunity and provide a mechanistic target for optimising rAd immunogenicity. http://dx.doi.org/10.1016/j.cyto.2014.07.159
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lower inflammation scores in the stomach compared to vaccinated wild-type mice. Furthermore, in vivo neutralization of IL-17A in sublingually vaccinated IFNc / mice totally abrogated protection against H. pylori infection. We next examined the mechanisms for induction and maintenance of IL 17A after SL vaccination by studying the role of cytokines IL 1b and IL-23 using gene knockout mice either lacking IL-1 signaling or IL-23 production respectively. Our results show that after SL vaccination with / H. pylori antigens and cholera toxin, IL-23p19 mice, but not IL-1RI / mice were protected against H. pylori infection. Gastric IL-17A responses could not be induced after challenge in the absence of IL-1 signaling, but could be maintained in the absence of IL-23. In summary, we report that IL-17A is important in reducing the bacterial load on the stomach of vaccinated mice which is dependent on intact IL-1 signaling, while IFNc may promote inflammation. Based on our results, mechanisms of vaccine-induced protection against H. pylori infection and the role of specific cytokines will be discussed. http://dx.doi.org/10.1016/j.cyto.2014.07.161
153 The effect of Gelam honey on the oxidative stress induced inflammatory pathways in pancreatic hamster cells
155 STAT5 regulation of TOX: Its dependence on IL-2 and role in cancer
Rajes Qvist, University of Malaya, Kuala Lumpur, Malaysia Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic b cell dysfunction which could result from glucose toxicity, inflammatory cytokines and oxidative stress. In the present study we investigated the effect of gelam honey and quercetin, on the oxidative stress induced inflammatory pathways and the proinflammatory cytokines. Methods: HIT-T15 cells were cultured in 5% CO2 and then preincubated with gelam honey extract (20, 40, 60 and 80 lg/ml., as well as quercetin (20,40, 60 and 80 lM), prior to stimulation by 20 and 50 mM glucose. Cell lysates were collected to determine the effect of honey extracts and quercetin on JNK and IKkb inflammatory pathways and the proinflammatory cytokines TNFa, IL-6 and IL-1b. Results: HIT-T15 cells cultured under hyperglycemic condition showed a significant increase in the inflammatory pathways by phosphorylating JNK, IKkb and IRS1at ser 307 (p < 0.05). At the same time there was a significant decrease in the phosphorylation AKt at ser 473 (p < 0.05). Pretreatment with gelam honey and quercetin reduced the expression of phosphorylated JNK, IKkb and IRS-1, thereby significantly reducing the expression of proinflammatory cytokines like TNFa, IL-6 and IL-1b (p < 0.05). At the same time there was a significant increase in the phosphorylated Akt showing the protective effects against inflammation and insulin resistance (p < 0.05). In conclusion our data suggest the potential use of the extract from gelam honey and quercetin in modulating the inflammation induced insulin signaling pathways. It is also possible that JNK and IKkb pathways could be a therapeutic target for Type 2 diabetes. http://dx.doi.org/10.1016/j.cyto.2014.07.160
154 The role of IL-17A and IFNc in vaccine-induced protection against Helicobacter pylori infection Louise Sjökvist-Ottsjö 1, Carl-Fredrik Flach 1, Staffan Nilsson 2, Rene de Waal Malefyt 3, Anna Walduck 4, Sukanya Raghavan 1, 1 Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden, 2 Department of Mathematical Sciences, Chalmers University of Technology, Gothenburg, Sweden, 3 Department of Immunology, Merck Research Laboratories, Palo Alto, CA, USA, 4 School of Applied Sciences, RMIT University, Bandoora, Victoria, Australia The aim of the project was to evaluate mechanisms of vaccine-induced protection against Helicobacter pylori infection in mice. In particular, to elucidate the role of cytokines induced by H. pylori infection in promoting the protective or pathogenic immune responses in the stomach. Our group has previously shown that sublingual (SL; under the tongue) vaccination with H. pylori antigens and cholera toxin as an adjuvant was efficient in reducing the bacterial load in the stomach of mice with enhanced IFNc and IL-17A responses in the stomach compared to unvaccinated mice. Using gene knockout mice and neutralizing antibodies, the impact of cytokines IFNc and IL-17A on the bacterial load, immune responses and gastric inflammation was addressed. We report that after SL vaccination, IFNc gene knockout (IFNc / ) mice were protected against H. pylori infection and had elevated IL-17A production and
Aradhana Rani 1, John Murphy 2, Stipo Jurcevic 1, 1 King’s College London, London, United Kingdom, 2 School of Life Sciences, University of Westminster, London, United Kingdom The Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway represents a rapid membrane to nucleus signaling system used by cytokines, hormones and growth factors. STAT5 is one of the seven STATs and is a key signaling protein which binds to chromatin at IFN-c activation site (GAS motifs) and leads to the activation or repression of target genes involved in cell differentiation, proliferation and survival. In this study, several genes directly regulated by STAT5 were identified using chromatin immunoprecipitation in human CD8 + T lymphocytes. Mapping of the genomic fragments revealed that approximately 50% of the target sites were either in the proximal promoter region or introns of genes. Of the 183 STAT5 responsive binding sites identified in CD8 + T cells, almost a fourth contained the canonical GAS motif. Bioinformatic analyses, identified several target-genes whose aberrant functions are associated with malignant transformation of cells, consistent with the frequent dysregulation of STAT5 noted in various cancers. We have identified two other genes regulated by STAT5 and an interesting gene gene identified here is TOX: which is a differentiation program of developing T-cells [1,2]. The STAT5 binding site identified here, mapped to an internal intron within the TOX gene. The histone methylation marks too were indicative of an active enhancer region. Mining for information about promoter/TSS on the UCSC genome browser, revealed the presence of a typical GAS motif. An upregulation of TOX was observed in activated CD8 + T cells upon stimulation with IL-2. Although its role is T cell development is well studied, its role in cancers is less known. Various studies have shown the association between TOX subfamily of genes and cancer [3,4]. Due to its role in cellular proliferation and differentiation, TOX could have implications in cancer therapeutics. In conclusion, this study identified a role for STAT5 in the regulation of TOX in human T lymphocytes. Its role in cancer is under investigation. TOX (Thymocyte Selection-Associated High Mobility Group Box).
References [1] Rani A, Greenlaw R, Runglall M, Jurcevic S, John S. FRA2 is a STAT5 target gene regulated by IL-2 in human CD4 T cells. PLoS One 2014;9:e90370. [2] Wilkinson B, Chen JY, Han P, Rufner KM, Goularte OD, et al.. TOX: an HMG box protein implicated in the regulation of thymocyte selection. Nat Immunol 2002;3:272–80. [3] Aliahmad P, Kadavallore A, de la Torre B, Kappes D, Kaye J. TOX is required for development of the CD4 T cell lineage gene program. J Immunol 2011;187:5931–40. [4] Rani A, Afzali B, Kelly A, Tewolde-Berhan L, Hackett M, et al.. IL-2 regulates expression of C-MAF in human CD4 T cells. J Immunol 2011;187:3721–9. http://dx.doi.org/10.1016/j.cyto.2014.07.162
Results: While the highest CD8 T cell responses were observed after vaccination with rAd vectors with high expression of antigen, the level of antigen expression inversely correlated with innate immune responses. Upon interrogation of candidate innate genes, augmenting type I IFN signaling decreased antigen expression while abrogating type I IFN signaling increased antigen expression and promoted early CD8 T cell expansion. More specifically and upstream of type I IFN induction, deficiency in STING but not RLR signaling pathways increased antigen expression and CD8 T cell expansion. Conclusions: These data illustrate a reciprocal relationship between antigen expression and innate immune responses for CD8 T cell immunity and provide a mechanistic target for optimising rAd immunogenicity. http://dx.doi.org/10.1016/j.cyto.2014.07.159
65
lower inflammation scores in the stomach compared to vaccinated wild-type mice. Furthermore, in vivo neutralization of IL-17A in sublingually vaccinated IFNc / mice totally abrogated protection against H. pylori infection. We next examined the mechanisms for induction and maintenance of IL 17A after SL vaccination by studying the role of cytokines IL 1b and IL-23 using gene knockout mice either lacking IL-1 signaling or IL-23 production respectively. Our results show that after SL vaccination with / H. pylori antigens and cholera toxin, IL-23p19 mice, but not IL-1RI / mice were protected against H. pylori infection. Gastric IL-17A responses could not be induced after challenge in the absence of IL-1 signaling, but could be maintained in the absence of IL-23. In summary, we report that IL-17A is important in reducing the bacterial load on the stomach of vaccinated mice which is dependent on intact IL-1 signaling, while IFNc may promote inflammation. Based on our results, mechanisms of vaccine-induced protection against H. pylori infection and the role of specific cytokines will be discussed. http://dx.doi.org/10.1016/j.cyto.2014.07.161
153 The effect of Gelam honey on the oxidative stress induced inflammatory pathways in pancreatic hamster cells
155 STAT5 regulation of TOX: Its dependence on IL-2 and role in cancer
Rajes Qvist, University of Malaya, Kuala Lumpur, Malaysia Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic b cell dysfunction which could result from glucose toxicity, inflammatory cytokines and oxidative stress. In the present study we investigated the effect of gelam honey and quercetin, on the oxidative stress induced inflammatory pathways and the proinflammatory cytokines. Methods: HIT-T15 cells were cultured in 5% CO2 and then preincubated with gelam honey extract (20, 40, 60 and 80 lg/ml., as well as quercetin (20,40, 60 and 80 lM), prior to stimulation by 20 and 50 mM glucose. Cell lysates were collected to determine the effect of honey extracts and quercetin on JNK and IKkb inflammatory pathways and the proinflammatory cytokines TNFa, IL-6 and IL-1b. Results: HIT-T15 cells cultured under hyperglycemic condition showed a significant increase in the inflammatory pathways by phosphorylating JNK, IKkb and IRS1at ser 307 (p < 0.05). At the same time there was a significant decrease in the phosphorylation AKt at ser 473 (p < 0.05). Pretreatment with gelam honey and quercetin reduced the expression of phosphorylated JNK, IKkb and IRS-1, thereby significantly reducing the expression of proinflammatory cytokines like TNFa, IL-6 and IL-1b (p < 0.05). At the same time there was a significant increase in the phosphorylated Akt showing the protective effects against inflammation and insulin resistance (p < 0.05). In conclusion our data suggest the potential use of the extract from gelam honey and quercetin in modulating the inflammation induced insulin signaling pathways. It is also possible that JNK and IKkb pathways could be a therapeutic target for Type 2 diabetes. http://dx.doi.org/10.1016/j.cyto.2014.07.160
154 The role of IL-17A and IFNc in vaccine-induced protection against Helicobacter pylori infection Louise Sjökvist-Ottsjö 1, Carl-Fredrik Flach 1, Staffan Nilsson 2, Rene de Waal Malefyt 3, Anna Walduck 4, Sukanya Raghavan 1, 1 Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden, 2 Department of Mathematical Sciences, Chalmers University of Technology, Gothenburg, Sweden, 3 Department of Immunology, Merck Research Laboratories, Palo Alto, CA, USA, 4 School of Applied Sciences, RMIT University, Bandoora, Victoria, Australia The aim of the project was to evaluate mechanisms of vaccine-induced protection against Helicobacter pylori infection in mice. In particular, to elucidate the role of cytokines induced by H. pylori infection in promoting the protective or pathogenic immune responses in the stomach. Our group has previously shown that sublingual (SL; under the tongue) vaccination with H. pylori antigens and cholera toxin as an adjuvant was efficient in reducing the bacterial load in the stomach of mice with enhanced IFNc and IL-17A responses in the stomach compared to unvaccinated mice. Using gene knockout mice and neutralizing antibodies, the impact of cytokines IFNc and IL-17A on the bacterial load, immune responses and gastric inflammation was addressed. We report that after SL vaccination, IFNc gene knockout (IFNc / ) mice were protected against H. pylori infection and had elevated IL-17A production and
Aradhana Rani 1, John Murphy 2, Stipo Jurcevic 1, 1 King’s College London, London, United Kingdom, 2 School of Life Sciences, University of Westminster, London, United Kingdom The Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway represents a rapid membrane to nucleus signaling system used by cytokines, hormones and growth factors. STAT5 is one of the seven STATs and is a key signaling protein which binds to chromatin at IFN-c activation site (GAS motifs) and leads to the activation or repression of target genes involved in cell differentiation, proliferation and survival. In this study, several genes directly regulated by STAT5 were identified using chromatin immunoprecipitation in human CD8 + T lymphocytes. Mapping of the genomic fragments revealed that approximately 50% of the target sites were either in the proximal promoter region or introns of genes. Of the 183 STAT5 responsive binding sites identified in CD8 + T cells, almost a fourth contained the canonical GAS motif. Bioinformatic analyses, identified several target-genes whose aberrant functions are associated with malignant transformation of cells, consistent with the frequent dysregulation of STAT5 noted in various cancers. We have identified two other genes regulated by STAT5 and an interesting gene gene identified here is TOX: which is a differentiation program of developing T-cells [1,2]. The STAT5 binding site identified here, mapped to an internal intron within the TOX gene. The histone methylation marks too were indicative of an active enhancer region. Mining for information about promoter/TSS on the UCSC genome browser, revealed the presence of a typical GAS motif. An upregulation of TOX was observed in activated CD8 + T cells upon stimulation with IL-2. Although its role is T cell development is well studied, its role in cancers is less known. Various studies have shown the association between TOX subfamily of genes and cancer [3,4]. Due to its role in cellular proliferation and differentiation, TOX could have implications in cancer therapeutics. In conclusion, this study identified a role for STAT5 in the regulation of TOX in human T lymphocytes. Its role in cancer is under investigation. TOX (Thymocyte Selection-Associated High Mobility Group Box).
References [1] Rani A, Greenlaw R, Runglall M, Jurcevic S, John S. FRA2 is a STAT5 target gene regulated by IL-2 in human CD4 T cells. PLoS One 2014;9:e90370. [2] Wilkinson B, Chen JY, Han P, Rufner KM, Goularte OD, et al.. TOX: an HMG box protein implicated in the regulation of thymocyte selection. Nat Immunol 2002;3:272–80. [3] Aliahmad P, Kadavallore A, de la Torre B, Kappes D, Kaye J. TOX is required for development of the CD4 T cell lineage gene program. J Immunol 2011;187:5931–40. [4] Rani A, Afzali B, Kelly A, Tewolde-Berhan L, Hackett M, et al.. IL-2 regulates expression of C-MAF in human CD4 T cells. J Immunol 2011;187:3721–9. http://dx.doi.org/10.1016/j.cyto.2014.07.162