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SS11-2 ISG15 and protein ISGylation
Cytokine 52 (2010) 80–81
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Cytokines and Infectious Diseases SS11-1 Therapeutic exploitation of shared IFNc and IFNa signaling for mycobacterial disease H. Bax, A.F. Freeman, A.P. Hsu, S.K. Browne, B.E. Marciano, M. Paulson, C. Fowler, L. Ding, E.P. Sampaio, S.M. Holland, Laboratory of Clinical Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA How interferon gamma (IFNc) leads to the control and killing of intracellular mycobacteria is still opaque. Interferon gamma receptors 1 and 2 (IFNcR1 and IFNcR2) as well as signal transducer and mediator of transcription 1 (STAT1) are necessary, as proven by the existence of specific mutations in these genes leading to severe, disseminated nontuberculous mycobacterial disease, which is typically fatal in childhood. Complete defects in IFNcR1 and IFNcR2 are especially severe due to loss of ability to activate STAT1 through the IFNcR complex. Faced with patients with complete IFNcR defects with severe disease we sought ways to bypass their defects. We reasoned that the interferon alpha receptor (IFNaR) also led to the activation of STAT1 and an overlapping set of gene targets. Therefore, we set out to evaluate the ability of IFNa to activate STAT1 and typically IFNc-responsive genes in the setting of partial and complete IFNcR deficiency. We confirmed that IFNa activates many of the same targets as IFNc, even in the setting of IFNcR deficiency, as was expected. We then used IFNa in the treatment of patients with IFNgR deficiency, monitoring in vivo and ex vivo effects. Pegylated IFNa induced both typical IFNa-induced genes, including MX1, IsG15 and CXCL11, as well as robustly induced more typically IFNcinduced genes IP-10, CXCL9, and IDO. More importantly, one patient with complete IFNcR1 deficiency and widely disseminated M. avium complex infection had a complete response to pegylated IFNa with disappearance of extensive pulmonary and mediastinal disease. One other patient had similar ex vivo responses and even though the treated patients presented with variable clinical responses, all had reduction in the extent of clinical disease and infection burden. The ability to bypass a severe defect in one cytokine receptor by pharmacologically exploiting redundancies in signaling is a novel approach to the treatment of cytokine defects.
doi:10.1016/j.cyto.2010.07.332
SS11-2 ISG15 and protein ISGylation Xiuli Cong, Brian Reuter, Ming Yan, Dong-Er Zhang, Department of Pathology, Division of Biological Sciences, and Moores UCSD Cancer Center, University of California San Diego, La Jolla, CA 92093, USA The ISG15 gene is strongly upregulated by type I interferon. It is expressed in response to a variety of stress conditions, especially during pathogen infections. The ISG15 protein belongs to the ubiquitin like protein family and is present in its free form or conjugated to hundreds of cellular proteins. Most of the currently known enzymes involved in protein ISG15 modification (ISGylation) are also encoded by interferon inducible genes, indicating that ISGylation is a tightly regulated process. Blood cells are important for both innate and adaptive immune responses. Following bone marrow transplantation, donor hematopoietic stem cells are recruited from their quiescent state to promote the rapid reconstitution in recipients. This dynamic process is tightly regulated by a complex of internal and external signals. We observed higher levels of Type I IFNs and protein ISGylation in the bone marrow of recipients shortly after transplantation. In order to clarify the physiological function of protein ISGylation, we used a mouse model that lacks protein ISGylation due to deficiency of ISG15 conjugating enzyme Ube1L (Ube1L / ). We analyzed the hematopoietic system in Ube1L / mice in steady-state hematopoiesis and its potential protective role during bone marrow reconstitution. Although steady-state hematopoiesis was unperturbed, transplantation experiment revealed a 50% reduction in repopulation potential of Ube1L-deficient cells at 3 weeks posttransplantation, but no differences at 6 and 12 weeks. A competitive transplantation experiment magnified and
extended this phenotype. Cell cycle analysis revealed that under the condition with high levels of IFNs and protein ISGylation, the Ube1L deficiency can cause G2/M phase block of cell cycle in hematopoietic multipotential progenitors. These observations indicate that although protein ISGylation is dispensable for steady-state hematopoiesis, it plays a significant role during interferon related stress response. doi:10.1016/j.cyto.2010.07.333
SS11-3 Interleukin-15 expression in dendritic cell and T cell subsets revealed using a novel reporter system Sara L. Colpitts 1, Thomas A. Stokalsek 1, Joshua, J. Obar 1, Caiying Guo 2, Leo Lefrançois 1, 1 Department of Immunology, Center for Integrated Immunology and Vaccine Research, 2 The Gene Targeting and Transgenic Facility, University of Connecticut Health Center, Farmington, CT, USA Interleukin-15 (IL-15) plays a multifaceted role in immune homeostasis, but the inability to detect IL-15 has stymied exploration of IL-15 biology in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing Emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in many cells including dendritic cells (DCs), macrophages, basophils and neutrophils. Individual DC subsets expressed varying levels of EmGFP/IL-15 depending on their phenotype and anatomical location. In the spleen, CD8+ DCs expressed significantly higher levels of EmGFP/IL-15 than CD8 DCs. However, the CD11cintMHCIIhigh migratory DCs in the lymph nodes (LNs) expressed the highest levels of EmGFP/IL-15 of any DC subset during steady state. When mice were infected with vesicular stomatitis virus, both CD8+ and CD8 DCs significantly upregulated EmGFP/IL-15 expression. Also, while both DC subsets exhibited type I interferon (IFN)-dependent mechanisms of EmGFP/IL-15 upregulation, only CD8 DCs used an IFN-independent mechanism to maintain elevated EmGFP/IL-15 expression over uninfected controls. To address the differential expression of EmGFP/ IL-15 within the DC lineage, we isolated hematopoietic progenitors in the bone marrow revealing high EmGFP/IL-15 expression in LSK (Lin Sca-1+c-kit+) cells that progressively waned as cells matured towards a DC fate. EmGFP/IL-15-expressing pre-DCs generated both EmGFP/IL-15int CD8+ and EmGFP/IL-15low CD8 DCs, suggesting regulated control of IL-15 expression throughout DC development. Analogous to DCs, downregulation of EmGFP/IL-15 also occurred during T cell development as early thymic progenitors progressed through the double negative stages in the thymus yielding EmGFP/IL-15neg ab T cells and EmGFP/IL-15low gd T cells in the periphery. A small subset of gd T cells found only in the peripheral LNs expressed high levels of EmGFP/IL-15 and largely corresponded to the naturally occurring CD44highCD62LlowCD27lowCCR6high IL-17-producing population. These findings provide evidence of previously unappreciated differences in IL-15 production between individual DC and T cell subsets during development, homeostasis, and following viral infection. doi:10.1016/j.cyto.2010.07.334
SS11-4 Local manipulation of IL-6 trans-signaling therapeutically enhances anti-microbial host defense Barbara Coles, Chantal Colmont, Ceri A. Fielding, Ann Kift-Morgan, Emily Hams, Nicholas Topley, Simon A. Jones, Department of Infection, Immunity & Biochemistry, The School of Medicine, Cardiff University, Wales, UK Antibiotic therapies have remained essentially unchanged for decades. Although this has resulted in the treatment of most common infections, the increased incidence of resistance (e.g., MRSA, VRSA) and lack of new antibiotics has led to a need for novel therapeutic approaches. One attractive solution is the use of biologics that enhance or
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Cytokines and Infectious Diseases SS11-1 Therapeutic exploitation of shared IFNc and IFNa signaling for mycobacterial disease H. Bax, A.F. Freeman, A.P. Hsu, S.K. Browne, B.E. Marciano, M. Paulson, C. Fowler, L. Ding, E.P. Sampaio, S.M. Holland, Laboratory of Clinical Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA How interferon gamma (IFNc) leads to the control and killing of intracellular mycobacteria is still opaque. Interferon gamma receptors 1 and 2 (IFNcR1 and IFNcR2) as well as signal transducer and mediator of transcription 1 (STAT1) are necessary, as proven by the existence of specific mutations in these genes leading to severe, disseminated nontuberculous mycobacterial disease, which is typically fatal in childhood. Complete defects in IFNcR1 and IFNcR2 are especially severe due to loss of ability to activate STAT1 through the IFNcR complex. Faced with patients with complete IFNcR defects with severe disease we sought ways to bypass their defects. We reasoned that the interferon alpha receptor (IFNaR) also led to the activation of STAT1 and an overlapping set of gene targets. Therefore, we set out to evaluate the ability of IFNa to activate STAT1 and typically IFNc-responsive genes in the setting of partial and complete IFNcR deficiency. We confirmed that IFNa activates many of the same targets as IFNc, even in the setting of IFNcR deficiency, as was expected. We then used IFNa in the treatment of patients with IFNgR deficiency, monitoring in vivo and ex vivo effects. Pegylated IFNa induced both typical IFNa-induced genes, including MX1, IsG15 and CXCL11, as well as robustly induced more typically IFNcinduced genes IP-10, CXCL9, and IDO. More importantly, one patient with complete IFNcR1 deficiency and widely disseminated M. avium complex infection had a complete response to pegylated IFNa with disappearance of extensive pulmonary and mediastinal disease. One other patient had similar ex vivo responses and even though the treated patients presented with variable clinical responses, all had reduction in the extent of clinical disease and infection burden. The ability to bypass a severe defect in one cytokine receptor by pharmacologically exploiting redundancies in signaling is a novel approach to the treatment of cytokine defects.
doi:10.1016/j.cyto.2010.07.332
SS11-2 ISG15 and protein ISGylation Xiuli Cong, Brian Reuter, Ming Yan, Dong-Er Zhang, Department of Pathology, Division of Biological Sciences, and Moores UCSD Cancer Center, University of California San Diego, La Jolla, CA 92093, USA The ISG15 gene is strongly upregulated by type I interferon. It is expressed in response to a variety of stress conditions, especially during pathogen infections. The ISG15 protein belongs to the ubiquitin like protein family and is present in its free form or conjugated to hundreds of cellular proteins. Most of the currently known enzymes involved in protein ISG15 modification (ISGylation) are also encoded by interferon inducible genes, indicating that ISGylation is a tightly regulated process. Blood cells are important for both innate and adaptive immune responses. Following bone marrow transplantation, donor hematopoietic stem cells are recruited from their quiescent state to promote the rapid reconstitution in recipients. This dynamic process is tightly regulated by a complex of internal and external signals. We observed higher levels of Type I IFNs and protein ISGylation in the bone marrow of recipients shortly after transplantation. In order to clarify the physiological function of protein ISGylation, we used a mouse model that lacks protein ISGylation due to deficiency of ISG15 conjugating enzyme Ube1L (Ube1L / ). We analyzed the hematopoietic system in Ube1L / mice in steady-state hematopoiesis and its potential protective role during bone marrow reconstitution. Although steady-state hematopoiesis was unperturbed, transplantation experiment revealed a 50% reduction in repopulation potential of Ube1L-deficient cells at 3 weeks posttransplantation, but no differences at 6 and 12 weeks. A competitive transplantation experiment magnified and
extended this phenotype. Cell cycle analysis revealed that under the condition with high levels of IFNs and protein ISGylation, the Ube1L deficiency can cause G2/M phase block of cell cycle in hematopoietic multipotential progenitors. These observations indicate that although protein ISGylation is dispensable for steady-state hematopoiesis, it plays a significant role during interferon related stress response. doi:10.1016/j.cyto.2010.07.333
SS11-3 Interleukin-15 expression in dendritic cell and T cell subsets revealed using a novel reporter system Sara L. Colpitts 1, Thomas A. Stokalsek 1, Joshua, J. Obar 1, Caiying Guo 2, Leo Lefrançois 1, 1 Department of Immunology, Center for Integrated Immunology and Vaccine Research, 2 The Gene Targeting and Transgenic Facility, University of Connecticut Health Center, Farmington, CT, USA Interleukin-15 (IL-15) plays a multifaceted role in immune homeostasis, but the inability to detect IL-15 has stymied exploration of IL-15 biology in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing Emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in many cells including dendritic cells (DCs), macrophages, basophils and neutrophils. Individual DC subsets expressed varying levels of EmGFP/IL-15 depending on their phenotype and anatomical location. In the spleen, CD8+ DCs expressed significantly higher levels of EmGFP/IL-15 than CD8 DCs. However, the CD11cintMHCIIhigh migratory DCs in the lymph nodes (LNs) expressed the highest levels of EmGFP/IL-15 of any DC subset during steady state. When mice were infected with vesicular stomatitis virus, both CD8+ and CD8 DCs significantly upregulated EmGFP/IL-15 expression. Also, while both DC subsets exhibited type I interferon (IFN)-dependent mechanisms of EmGFP/IL-15 upregulation, only CD8 DCs used an IFN-independent mechanism to maintain elevated EmGFP/IL-15 expression over uninfected controls. To address the differential expression of EmGFP/ IL-15 within the DC lineage, we isolated hematopoietic progenitors in the bone marrow revealing high EmGFP/IL-15 expression in LSK (Lin Sca-1+c-kit+) cells that progressively waned as cells matured towards a DC fate. EmGFP/IL-15-expressing pre-DCs generated both EmGFP/IL-15int CD8+ and EmGFP/IL-15low CD8 DCs, suggesting regulated control of IL-15 expression throughout DC development. Analogous to DCs, downregulation of EmGFP/IL-15 also occurred during T cell development as early thymic progenitors progressed through the double negative stages in the thymus yielding EmGFP/IL-15neg ab T cells and EmGFP/IL-15low gd T cells in the periphery. A small subset of gd T cells found only in the peripheral LNs expressed high levels of EmGFP/IL-15 and largely corresponded to the naturally occurring CD44highCD62LlowCD27lowCCR6high IL-17-producing population. These findings provide evidence of previously unappreciated differences in IL-15 production between individual DC and T cell subsets during development, homeostasis, and following viral infection. doi:10.1016/j.cyto.2010.07.334
SS11-4 Local manipulation of IL-6 trans-signaling therapeutically enhances anti-microbial host defense Barbara Coles, Chantal Colmont, Ceri A. Fielding, Ann Kift-Morgan, Emily Hams, Nicholas Topley, Simon A. Jones, Department of Infection, Immunity & Biochemistry, The School of Medicine, Cardiff University, Wales, UK Antibiotic therapies have remained essentially unchanged for decades. Although this has resulted in the treatment of most common infections, the increased incidence of resistance (e.g., MRSA, VRSA) and lack of new antibiotics has led to a need for novel therapeutic approaches. One attractive solution is the use of biologics that enhance or