- Email: [email protected]
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Abstract / Cytokine 70 (2014) 28–79
compared with WT cells. Our results indicate that Nod1 cooperates with Nod2 or TLRs to produce cytokines in macrophages in response to M. tuberculosis.
111 Aryl hydrocarbon receptor negatively regulates type I interferon production and the development of murine lupus
http://dx.doi.org/10.1016/j.cyto.2014.07.115 Soyoung Lee, Barry Ripley, Ichino Chinen, David Millrine, Tadamitsu Kishimoto, Immune Regulation, Immunology Frontier Research Center, Osaka University, Osaka, Japan 109 Granulocyte macrophage-colony stimulating factor and interleukin-3 are not essential for endotoxin-induced extramedullary myelopoiesis Ming-Chin Lee, Andrew D. Cook, Derek C. Lacey, John A. Hamilton, The University of Melbourne, Melbourne, VIC, Australia Several studies have reported that splenic extramedullary haematopoiesis is important during certain inflammatory conditions. The haematopoietic growth factor, granulocyte macrophage-colony stimulating factor (GM-CSF), is known to stimulate the growth of in vitro murine macrophages from bone marrow progenitors. Current literature suggests that GM-CSF and interleukin-3 (IL-3) are implicated in regulating the survival and proliferative activity of granulocyte-monocyte progenitors (GMPs) during atherosclerosis. In the current study, we sought to determine the involvement of GM-CSF and/or IL-3 in splenic haematopoiesis following multiple intra-peritoneal (i.p) injections of endotoxin, lipopolysaccharide (LPS). GM-CSF / mice and C57BL/6 wild-type (WT) mice were administered with 10 lg Escherichia coli LPS into the peritoneal cavity for four consecutive days. In experiments using neutralizing antibodies against GM-CSF and/or IL-3, 150 lg of the neutralizing antibody was administered on day 1 and 2. Spleen, bone marrow and blood were collected and analysed using flow cytometry. In response to endotoxin challenge, both GM-CSF / and WT mice displayed a comparable degree of splenomegaly, with increased total spleen cell numbers, mainly attributable to an increase in splenic myeloid lineage cells, including neutrophils and subsets of monocytes (Ly6Chi and Ly6Clo). The numbers of splenic GMPs were alsosignificantly increased following multiple endotoxin injections in both WT and GMCSF / mice. The cellular composition and number of bone marrow were unaltered following endotoxin challenge, whereas there was significant leukocytosis in peripheral blood of both WT and GM-CSF / mice. Neutralizing GM-CSF and/or IL-3 in WT mice, which received multiple endotoxin injections, failed to inhibit the development of splenomegaly, in which the numbers of GMPs were again increased amongst all treatment groups. Taken together, these results demonstrate that, while splenic extramedullary myelopoiesis may be important in supplying inflammatory cells during certain inflammatory models, it is independent of GM-CSF and IL-3 following multiple endotoxin challenges. http://dx.doi.org/10.1016/j.cyto.2014.07.116
110 SO-HW01, a medicinal plant extract, inhibits inflammasome activation and prevents endotoxin-induced septic shock in mice Sang-Myeong Lee, Dong-Won Seo, Young-Joo Yi, Da-hee Kim, Chonbuk National Universrity, Iksan, South Korea Inflammasome is known as a cytosolic protein complex of nucleotide-binding domain and leucine rich repeat containing protein (NLRs) or AIM2, ASC and caspase-1 and assembled in response to the detection of endogenous and pathogen-associated danger signals. Activated caspase-1 mediates the proteolytic maturation of interleukin-1b (IL-1b), which plays an important role in innate immune responses. However, dysregulated production of IL-1b is associated with a number of autoinflammatory disorders and its inhibition has been proved to be beneficial in the treatment of these diseases. The purpose of this study was to screen inflammasome inhibitors from medicinal herbal extracts. We identified that SO-HW01 possessed an inhibitory effect on inflammasome induction. SO-HW01 effectively suppressed IL-1b production triggered by ATP or nigericin or Listeria monocytogenes, which induce NLRP3 inflammasome, Salmonella thyphimurium, which involve NLRC4, and dsDNA, which active AIM2, in a dose dependent manner. The mechanism of action of SO-HW01 was related to the inhibition of the cleavage of pro-caspase-1 and proIL-1b which in turn suppressed the activation of inflammasome. SO-HW01 significantly reduced LPS-induced septic shock by inhibiting production of IL-1b and IL18. Taken together, our results demonstrate that HSO inhibits NLRP3, NLRC4, and AIM2 inflammasome activation and it has a potential use in the treatment of inflammsome-mediated diseases. http://dx.doi.org/10.1016/j.cyto.2014.07.117
Background: Systemic lupus erythematosus is a highly heterogenous multi-organ autoimmune disease characterized by the production of diverse autoantibodies and broad spectrum of clinical manifestations. Various studies suggest an important role for type I interferon (IFN) in the pathogenesis of lupus. Type I IFN signaling and subsequent feedback induction of type I IFN production, are critically dependent on the STAT1 transcription factor. However, how type I IFN activity is regulated through inhibition of STAT1 function is poorly understood. In our previous study we demonstrated that the aryl hydrocarbon receptor (AHR) inhibits STAT1 activity through TLR4 signaling. In the current study we investigated a role for AHR in type I IFN signaling through TLR7/9 pathways. Methods and results: We showed that type I IFN production and signaling both in plasmacytoid dendritic cells (pDCs), and in vivo, is significantly higher in AHR knockout mice compared to wild-type mice, following stimulation with TLR9 ligands. Type I IFN production was suppressed by AHR agonist and in AHR over-expressing cells. We demonstrated that through TLR9 signaling or by IFN-a stimulation in pDCs, expression of AHR is induced, which in-turn forms an inhibitory interaction with STAT1. This interaction attenuates both type I IFN signaling and in-turn, positive-feedback induction of type I IFN. Interestingly, we also found that AHR also forms an inhibitory interaction with IRF7 (a transcription factor which induces type I IFN production). In pristane-induced murine lupus, we found that expression of IFN-stimulated genes, accumulation of Ly6chigh IFN producing cells, production of type I IFN-dependent autoantibody and severity of proteinurea, were significantly higher in AHR knockout mice compared to wild-type mice. Conclusion: We firstly report that AHR negatively regulates type I IFN production and signaling through an inhibitory interaction with IRF7 and STAT1 respectively and inhibits interferon-dependent disease in murine lupus. http://dx.doi.org/10.1016/j.cyto.2014.07.118
112 The mechanism and application of a superagonistic antibody for human IL-21 Yew Ann Leong 1, Yanfang Cui 1, Zhian Chen 1, Fiona Wightman 2, Yaping Chen 1, Sharon Lewin 2, Alan Landay 3, Charles Mackay 1, Di Yu 1, 1 Immunology (Clayton), Monash University, Clayton, Vic, Australia, 2 Central Clinical School, Monash University, Prahran, Vic, Australia, 3 Immunology–Microbiology, Rush University, Chicago, IL, United States Aims: Interleukin-21 (IL-21) is a cytokine of common c chain family, predominantly produced by activated CD4 T cells. IL-21 has broad immuno-stimulatory properties required for antibody affinity maturation and memory formation, as well as for maintenance of cytotoxic immunity from exhaustion during chronic infection. Indeed, recombinant human IL-21 (hIL-21) has shown great promise in phase I and II clinical trials to boost immunity in cancer immunotherapies. We took a novel approach to develop a unique agonistic antibody, 2P2 to hIL-21, which enhances bioactivity of hIL-21 to 10–100 folds in vitro and significantly prolonged half-life of hIL-21 in vivo. This project aims to identify the mechanism of the super-agonistic activityof 2P2 using bio-assays and crystallography. In addition, the application of anti-infection and anti-tumour functions of this super-agonistic antibody will be tested in human cell culture and in vivo mouse models. Methods: Mechanisms of 2P2 superagonistic property will be studied by epitope mapping and crystallography. To investigate the in vivo superagonistic activity of 2P2, human IL-21R (hIL-21R) knock in mice have been generated by replacing (knock-in) the endogenous mouse IL-21R with hIL-21R. These hIL21R KI mice are being used to determine the half-life of 2P2/IL-21 and to test the activity of 2P2 in anti-infection and anti-tumour models. To investigate therapeutic applicability of 2P2, anti-HIV activity in the peripheral blood mononuclear cells (PBMC) from HIV infected patients will be determined in the presence of hIL-21/2P2. Results and conclusions: 2P2 binds to the epitope proximate to hIL-21/hIL-21R binding interface and the structural analysis suggests it might stabilize the ligand-receptor binding. Functionally, 2P2 has been shown to prolong the half-life of IL-21 and promote cytotoxicity in vivo. In parallel, 2P2 is able to enhance the cytotoxicity of NK cells and CD8 T cells in PBMCs from HIV-infection patients. All together, this study reveals a new class of superagonistic antibody with broad application potential. http://dx.doi.org/10.1016/j.cyto.2014.07.119
Abstract / Cytokine 70 (2014) 28–79
compared with WT cells. Our results indicate that Nod1 cooperates with Nod2 or TLRs to produce cytokines in macrophages in response to M. tuberculosis.
111 Aryl hydrocarbon receptor negatively regulates type I interferon production and the development of murine lupus
http://dx.doi.org/10.1016/j.cyto.2014.07.115 Soyoung Lee, Barry Ripley, Ichino Chinen, David Millrine, Tadamitsu Kishimoto, Immune Regulation, Immunology Frontier Research Center, Osaka University, Osaka, Japan 109 Granulocyte macrophage-colony stimulating factor and interleukin-3 are not essential for endotoxin-induced extramedullary myelopoiesis Ming-Chin Lee, Andrew D. Cook, Derek C. Lacey, John A. Hamilton, The University of Melbourne, Melbourne, VIC, Australia Several studies have reported that splenic extramedullary haematopoiesis is important during certain inflammatory conditions. The haematopoietic growth factor, granulocyte macrophage-colony stimulating factor (GM-CSF), is known to stimulate the growth of in vitro murine macrophages from bone marrow progenitors. Current literature suggests that GM-CSF and interleukin-3 (IL-3) are implicated in regulating the survival and proliferative activity of granulocyte-monocyte progenitors (GMPs) during atherosclerosis. In the current study, we sought to determine the involvement of GM-CSF and/or IL-3 in splenic haematopoiesis following multiple intra-peritoneal (i.p) injections of endotoxin, lipopolysaccharide (LPS). GM-CSF / mice and C57BL/6 wild-type (WT) mice were administered with 10 lg Escherichia coli LPS into the peritoneal cavity for four consecutive days. In experiments using neutralizing antibodies against GM-CSF and/or IL-3, 150 lg of the neutralizing antibody was administered on day 1 and 2. Spleen, bone marrow and blood were collected and analysed using flow cytometry. In response to endotoxin challenge, both GM-CSF / and WT mice displayed a comparable degree of splenomegaly, with increased total spleen cell numbers, mainly attributable to an increase in splenic myeloid lineage cells, including neutrophils and subsets of monocytes (Ly6Chi and Ly6Clo). The numbers of splenic GMPs were alsosignificantly increased following multiple endotoxin injections in both WT and GMCSF / mice. The cellular composition and number of bone marrow were unaltered following endotoxin challenge, whereas there was significant leukocytosis in peripheral blood of both WT and GM-CSF / mice. Neutralizing GM-CSF and/or IL-3 in WT mice, which received multiple endotoxin injections, failed to inhibit the development of splenomegaly, in which the numbers of GMPs were again increased amongst all treatment groups. Taken together, these results demonstrate that, while splenic extramedullary myelopoiesis may be important in supplying inflammatory cells during certain inflammatory models, it is independent of GM-CSF and IL-3 following multiple endotoxin challenges. http://dx.doi.org/10.1016/j.cyto.2014.07.116
110 SO-HW01, a medicinal plant extract, inhibits inflammasome activation and prevents endotoxin-induced septic shock in mice Sang-Myeong Lee, Dong-Won Seo, Young-Joo Yi, Da-hee Kim, Chonbuk National Universrity, Iksan, South Korea Inflammasome is known as a cytosolic protein complex of nucleotide-binding domain and leucine rich repeat containing protein (NLRs) or AIM2, ASC and caspase-1 and assembled in response to the detection of endogenous and pathogen-associated danger signals. Activated caspase-1 mediates the proteolytic maturation of interleukin-1b (IL-1b), which plays an important role in innate immune responses. However, dysregulated production of IL-1b is associated with a number of autoinflammatory disorders and its inhibition has been proved to be beneficial in the treatment of these diseases. The purpose of this study was to screen inflammasome inhibitors from medicinal herbal extracts. We identified that SO-HW01 possessed an inhibitory effect on inflammasome induction. SO-HW01 effectively suppressed IL-1b production triggered by ATP or nigericin or Listeria monocytogenes, which induce NLRP3 inflammasome, Salmonella thyphimurium, which involve NLRC4, and dsDNA, which active AIM2, in a dose dependent manner. The mechanism of action of SO-HW01 was related to the inhibition of the cleavage of pro-caspase-1 and proIL-1b which in turn suppressed the activation of inflammasome. SO-HW01 significantly reduced LPS-induced septic shock by inhibiting production of IL-1b and IL18. Taken together, our results demonstrate that HSO inhibits NLRP3, NLRC4, and AIM2 inflammasome activation and it has a potential use in the treatment of inflammsome-mediated diseases. http://dx.doi.org/10.1016/j.cyto.2014.07.117
Background: Systemic lupus erythematosus is a highly heterogenous multi-organ autoimmune disease characterized by the production of diverse autoantibodies and broad spectrum of clinical manifestations. Various studies suggest an important role for type I interferon (IFN) in the pathogenesis of lupus. Type I IFN signaling and subsequent feedback induction of type I IFN production, are critically dependent on the STAT1 transcription factor. However, how type I IFN activity is regulated through inhibition of STAT1 function is poorly understood. In our previous study we demonstrated that the aryl hydrocarbon receptor (AHR) inhibits STAT1 activity through TLR4 signaling. In the current study we investigated a role for AHR in type I IFN signaling through TLR7/9 pathways. Methods and results: We showed that type I IFN production and signaling both in plasmacytoid dendritic cells (pDCs), and in vivo, is significantly higher in AHR knockout mice compared to wild-type mice, following stimulation with TLR9 ligands. Type I IFN production was suppressed by AHR agonist and in AHR over-expressing cells. We demonstrated that through TLR9 signaling or by IFN-a stimulation in pDCs, expression of AHR is induced, which in-turn forms an inhibitory interaction with STAT1. This interaction attenuates both type I IFN signaling and in-turn, positive-feedback induction of type I IFN. Interestingly, we also found that AHR also forms an inhibitory interaction with IRF7 (a transcription factor which induces type I IFN production). In pristane-induced murine lupus, we found that expression of IFN-stimulated genes, accumulation of Ly6chigh IFN producing cells, production of type I IFN-dependent autoantibody and severity of proteinurea, were significantly higher in AHR knockout mice compared to wild-type mice. Conclusion: We firstly report that AHR negatively regulates type I IFN production and signaling through an inhibitory interaction with IRF7 and STAT1 respectively and inhibits interferon-dependent disease in murine lupus. http://dx.doi.org/10.1016/j.cyto.2014.07.118
112 The mechanism and application of a superagonistic antibody for human IL-21 Yew Ann Leong 1, Yanfang Cui 1, Zhian Chen 1, Fiona Wightman 2, Yaping Chen 1, Sharon Lewin 2, Alan Landay 3, Charles Mackay 1, Di Yu 1, 1 Immunology (Clayton), Monash University, Clayton, Vic, Australia, 2 Central Clinical School, Monash University, Prahran, Vic, Australia, 3 Immunology–Microbiology, Rush University, Chicago, IL, United States Aims: Interleukin-21 (IL-21) is a cytokine of common c chain family, predominantly produced by activated CD4 T cells. IL-21 has broad immuno-stimulatory properties required for antibody affinity maturation and memory formation, as well as for maintenance of cytotoxic immunity from exhaustion during chronic infection. Indeed, recombinant human IL-21 (hIL-21) has shown great promise in phase I and II clinical trials to boost immunity in cancer immunotherapies. We took a novel approach to develop a unique agonistic antibody, 2P2 to hIL-21, which enhances bioactivity of hIL-21 to 10–100 folds in vitro and significantly prolonged half-life of hIL-21 in vivo. This project aims to identify the mechanism of the super-agonistic activityof 2P2 using bio-assays and crystallography. In addition, the application of anti-infection and anti-tumour functions of this super-agonistic antibody will be tested in human cell culture and in vivo mouse models. Methods: Mechanisms of 2P2 superagonistic property will be studied by epitope mapping and crystallography. To investigate the in vivo superagonistic activity of 2P2, human IL-21R (hIL-21R) knock in mice have been generated by replacing (knock-in) the endogenous mouse IL-21R with hIL-21R. These hIL21R KI mice are being used to determine the half-life of 2P2/IL-21 and to test the activity of 2P2 in anti-infection and anti-tumour models. To investigate therapeutic applicability of 2P2, anti-HIV activity in the peripheral blood mononuclear cells (PBMC) from HIV infected patients will be determined in the presence of hIL-21/2P2. Results and conclusions: 2P2 binds to the epitope proximate to hIL-21/hIL-21R binding interface and the structural analysis suggests it might stabilize the ligand-receptor binding. Functionally, 2P2 has been shown to prolong the half-life of IL-21 and promote cytotoxicity in vivo. In parallel, 2P2 is able to enhance the cytotoxicity of NK cells and CD8 T cells in PBMCs from HIV-infection patients. All together, this study reveals a new class of superagonistic antibody with broad application potential. http://dx.doi.org/10.1016/j.cyto.2014.07.119