- Email: [email protected]
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Abstract / Cytokine 63 (2013) 243–314
inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitor LY-294002 (p 6 0.01). TGF-b1 induced TREM-2 mRNA and protein expression were significantly reduced by the p38 MAP kinase inhibitor SB203580 (p 6 0.001). The role of SMAD-3 and activating transcription factor 2 in this pathway are now being investigated. Interestingly, the ERK1/2 pathway inhibitor, PD98059 suppressed TGF-b1 induced TREM-2 expression at the protein level only, revealing an alternative pathway of regulation. To look at the functional effects of TREM-2 we measured the effect of TREM-2 siRNA knockdown on TGF-b1 induced IL-1b, IL-8 and matrix metalloproteinase 1 (MMP-1) mRNA in THP-1 cells. TREM-2 knockdown inhibited TGF-b1 induced MMP-1 mRNA by 73% (p 6 0.001) but had no effect on the increase in IL-1b and IL-8. In conclusion, we show that IL-4 induces TREM-2 via a STAT-6 independent signalling pathway involving PI3K and identified TGF-b1 as a novel inducer of TREM-2. TGF-b1 induces TREM-2 through two mechanisms requiring p38 and ERK1/2 signalling pathways. IL-4 and TGF-b1 may be involved in increased TREM-2 expression seen in inflammatory disease. We also identify a new role for TREM-2 in the regulation of MMP-1 which may be how TREM-2 exerts its protective effects in wound healing.
As expected, compared to mock transfected cells, RAW-IL-37 cells did not produce significant IL-6 after LPS stimulation. Pretreatment with anti-IL-37 did not affect the IL-6 response in RAW-IL-37 or control cells. Next, we pretreated IL-37tg and wild type C57/Bl6 mice with 200 lg of the anti-IL-37-IgG or control IgG 2 h prior to injecting 100 lg of LPS. After 4 and 24 h serum was prepared for IL-6 levels. Pretreatment with the anti-IL-37-IgG did not modulate the LPS response of wildtype C57/Bl6 mice. In contrast, there was a 2.5-fold increase of serum IL-6 after LPS administration in IL-37tg mice that had received anti-IL-37-IgG compared to control IgG. Thus neutralizing endogenous IL-37 in IL37tg mice abrogates the protective effect of IL-37 in a model of septic shock. We therefore conclude that LPS induces the release of biologically active IL-37 from IL-37tg mice in vivo and that IL-37 binds to membrane receptors resulting in a suppression of LPS-inducible genes. In contrast, intracellular IL-37, which is not targeted by anti-IL-37-IgG, has major impact upon LPS stimulation in transfected cells.
http://dx.doi.org/10.1016/j.cyto.2013.06.033 http://dx.doi.org/10.1016/j.cyto.2013.06.031
29 Impact of HSV-1 induced VEGF-A on corneal lymphangiogenesis during the development of herpetic stromal keratitis Katie M. Bryant-Hudson a, Daniel J.J. Carr a,b, a Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA, b Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Much of the research regarding lymphangiogenesis and disease has focused on lymphedema and cancer biology. There is still much to be elucidated regarding the impact of lymphatic vessel development during microbial pathogenesis. One function of lymphatic vessels is to drain soluble antigen and antigen-presenting cells from the periphery to the draining lymph nodes, facilitating the mobilization of an adaptive immune response. However, the resulting immune response can lead to detrimental tissue pathology. This is extremely important during ocular disease, wherein transparency of the tissues anterior to the retina is required for normal vision. Herpes simplex virus type-1 (HSV-1) infection of the cornea can result in herpetic stromal keratitis (HSK), an immunopathologic condition characterized by opacity, edema, and neovascularization of the cornea. The impact of hemangiogenesis during an HSV-1 ocular infection has been well studied; however, the impact of lymphangiogenesis in the cornea has only recently been investigated. Previous studies indicate HSV-1 induces lymphangiogenesis via the expression of VEGF-A by infected cornea epithelial cells during acute infection. Furthermore, blood and lymphatic vessels were found throughout the corneas of latently infected animals (day 30 postinfection (pi)). The goal of the current study is to identify the growth factors and cells that contribute to the maintenance and further development of lymphatic vessels in the cornea following the resolution of infection. Results indicate there is a substantial increase in LYVE-1-postive lymphatic vessels by day 14 pi, when HSK develops. Preliminary analysis of 20 growth factors and cytokines revealed robust expression of IL-6, IL-1b, HGF, MMP9, and VEGF-A preceding day 14 pi. It is anticipated that results from this study may lead to new treatment strategies to attenuate the development of HSK.
31 Type I IFNs enhance metabolic activation to enable an antiviral response Daniel J. Burke a, Leonidas C. Platanias b, Eleanor N. Fish a, a Toronto General Research Institute, University Health Network & Dept. of Immunology, University of Toronto, Canada, b Robert H. Lurie Comprehensive Cancer Center and Division of Hematology– Oncology, Feinberg School of Medicine, Northwestern University, Chicago, United States Type I IFNs induce an antiviral immune response within hours of virus infection, mediated by activation of signaling pathways downstream of the type I IFN receptor. An IFN-induced antiviral response requires both transcriptional activation of genes and the rapid translation of mRNAs into proteins that effect an antiviral response. Both these processes are energy taxing. Cognizant that IFNs activate both the PI3K-Akt/mTOR pathway that is associated with nutrient sensing, and AMPK, associated with stress-induced metabolic activation, we undertook studies to investigate whether IFN-alpha/beta treatment influences metabolic events to enable an antiviral response. Using a panel of mouse embryonic fibroblasts null for Akt1/2, p85ß, AMPKa, TSC2 or 4E-BP1, i.e. with defects in various signaling intermediates along the PI3K/mTOR pathway, we provide evidence for IFN-induced regulation of glucose uptake, mediated by activation of the PI3K/mTOR pathway. Notably, we demonstrate that IFN regulation of glucose uptake is required for an antiviral response to infection with the cardiotropic virus, coxsackievirus B3 (CVB3). In addition, we provide evidence for IFN regulated increases in ATP production. In vivo, we provide evidence that activation of AMPK by the pharmacological agent metformin enhances the antiviral effects of IFN-ß treatment in mice infected with CVB3, lending further support that metabolic activation enhances an IFN-induced antiviral response.
http://dx.doi.org/10.1016/j.cyto.2013.06.034
32 IRF7-dependent, type I IFN (IFN-I) production induces lethal immune-mediated disease in STAT1 KO mice infected with LCMV
http://dx.doi.org/10.1016/j.cyto.2013.06.032 Iain L. Campbell, Wen Li, Markus J. Hofer, School of Molecular Bioscience, University of Sydney, NSW, Australia 30 Anti-IL-37 antibody abrogates the protection in IL-37 transgenic mice to LPSinduced septic shock Ana-Maria Bulau a, Andrea Gruschka a, Rahel Schwaiger a, Charles A. Dinarello b, Philip Bufler a, a Department of Pediatrics, Dr. von Hauner Children’s Hospital, LudwigMaximilians-University, Munich, Germany, b University of Colorado Denver, Aurora, CO, USA IL-37 is a fundamental inhibitor of innate immunity by reducing systemic and local inflammation. For example, mice transgenic for the IL-37 precursor (IL-37tg) are protected against LPS-induced septic shock. We have observed that processing of the IL-37 precursor by caspase-1 is required for its anti-inflammatory function in vitro and that IL-37 is secreted into the extracellular space. In the present study, we intended to neutralize secreted IL-37 in transfected macrophage cells and in IL37tg mice in order to determine the functional role of extracellular IL-37. RAW macrophages expressing IL-37 (RAW-IL-37) and mock transfected cells were preincubated with goat anti-human IL-37-IgG (R&D Systems) or control IgG for 4 h and then stimulated with 100 ng/ml LPS for 18 h. Supernatants were analyzed for IL-6.
STAT1 KO but not WT, STAT2 KO, IRF9 KO or IFNAR KO mice succumb to a lethal CD4+ T cell-mediated disease following systemic infection with lymphocytic choriomeningitis virus (LCMV). In previous studies we showed: (i) LCMV infection induces IRF7 gene expression in a STAT1-independent, but STAT2-dependent manner, and (2) IRF7 is required for IFN-alpha but not IFN-besa production in LCMV infection. Here we examined the role of IRF7 in the lethal host response to LCMV infection in STAT1 KO mice. In contrast to STAT1 KO mice, STAT1/IRF7 double KO mice survived LCMV infection and had a marked reduction in immune pathology affecting key organs including the liver, kidney and lung. LCMV infection in STAT1 KO mice was associated with a significant elevation of a number of cytokines in the serum including IFNakpga, IFN-besa, IFN-calla, IL-5, IL-6 and MCP1 but this response was absent in STAT1/IRF7 KO mice which had a modest increase in MCP-1 only. In addition, in the spleen of LCMV-infected STAT1 KO mice, IFN-besa and IFN-calla mRNAs were also much more highly induced when compared with WT mice, while in STAT1/ IRF7 KO mice IFN-calla mRNA induction was similar to WT animals, there was no induction of IFN-besa mRNA. To elucidate further a possible role of IFN-I in lethal disease, we examined STAT1/IFNAR double KO mice. In contrast to STAT1 KO mice that all succumbed to lethal disease, STAT1/IFNAR KO mice all survived infection with LCMV. In conclusion, lethality resulting from LCMV infection in STAT1 KO mice
Abstract / Cytokine 63 (2013) 243–314
inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitor LY-294002 (p 6 0.01). TGF-b1 induced TREM-2 mRNA and protein expression were significantly reduced by the p38 MAP kinase inhibitor SB203580 (p 6 0.001). The role of SMAD-3 and activating transcription factor 2 in this pathway are now being investigated. Interestingly, the ERK1/2 pathway inhibitor, PD98059 suppressed TGF-b1 induced TREM-2 expression at the protein level only, revealing an alternative pathway of regulation. To look at the functional effects of TREM-2 we measured the effect of TREM-2 siRNA knockdown on TGF-b1 induced IL-1b, IL-8 and matrix metalloproteinase 1 (MMP-1) mRNA in THP-1 cells. TREM-2 knockdown inhibited TGF-b1 induced MMP-1 mRNA by 73% (p 6 0.001) but had no effect on the increase in IL-1b and IL-8. In conclusion, we show that IL-4 induces TREM-2 via a STAT-6 independent signalling pathway involving PI3K and identified TGF-b1 as a novel inducer of TREM-2. TGF-b1 induces TREM-2 through two mechanisms requiring p38 and ERK1/2 signalling pathways. IL-4 and TGF-b1 may be involved in increased TREM-2 expression seen in inflammatory disease. We also identify a new role for TREM-2 in the regulation of MMP-1 which may be how TREM-2 exerts its protective effects in wound healing.
As expected, compared to mock transfected cells, RAW-IL-37 cells did not produce significant IL-6 after LPS stimulation. Pretreatment with anti-IL-37 did not affect the IL-6 response in RAW-IL-37 or control cells. Next, we pretreated IL-37tg and wild type C57/Bl6 mice with 200 lg of the anti-IL-37-IgG or control IgG 2 h prior to injecting 100 lg of LPS. After 4 and 24 h serum was prepared for IL-6 levels. Pretreatment with the anti-IL-37-IgG did not modulate the LPS response of wildtype C57/Bl6 mice. In contrast, there was a 2.5-fold increase of serum IL-6 after LPS administration in IL-37tg mice that had received anti-IL-37-IgG compared to control IgG. Thus neutralizing endogenous IL-37 in IL37tg mice abrogates the protective effect of IL-37 in a model of septic shock. We therefore conclude that LPS induces the release of biologically active IL-37 from IL-37tg mice in vivo and that IL-37 binds to membrane receptors resulting in a suppression of LPS-inducible genes. In contrast, intracellular IL-37, which is not targeted by anti-IL-37-IgG, has major impact upon LPS stimulation in transfected cells.
http://dx.doi.org/10.1016/j.cyto.2013.06.033 http://dx.doi.org/10.1016/j.cyto.2013.06.031
29 Impact of HSV-1 induced VEGF-A on corneal lymphangiogenesis during the development of herpetic stromal keratitis Katie M. Bryant-Hudson a, Daniel J.J. Carr a,b, a Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA, b Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Much of the research regarding lymphangiogenesis and disease has focused on lymphedema and cancer biology. There is still much to be elucidated regarding the impact of lymphatic vessel development during microbial pathogenesis. One function of lymphatic vessels is to drain soluble antigen and antigen-presenting cells from the periphery to the draining lymph nodes, facilitating the mobilization of an adaptive immune response. However, the resulting immune response can lead to detrimental tissue pathology. This is extremely important during ocular disease, wherein transparency of the tissues anterior to the retina is required for normal vision. Herpes simplex virus type-1 (HSV-1) infection of the cornea can result in herpetic stromal keratitis (HSK), an immunopathologic condition characterized by opacity, edema, and neovascularization of the cornea. The impact of hemangiogenesis during an HSV-1 ocular infection has been well studied; however, the impact of lymphangiogenesis in the cornea has only recently been investigated. Previous studies indicate HSV-1 induces lymphangiogenesis via the expression of VEGF-A by infected cornea epithelial cells during acute infection. Furthermore, blood and lymphatic vessels were found throughout the corneas of latently infected animals (day 30 postinfection (pi)). The goal of the current study is to identify the growth factors and cells that contribute to the maintenance and further development of lymphatic vessels in the cornea following the resolution of infection. Results indicate there is a substantial increase in LYVE-1-postive lymphatic vessels by day 14 pi, when HSK develops. Preliminary analysis of 20 growth factors and cytokines revealed robust expression of IL-6, IL-1b, HGF, MMP9, and VEGF-A preceding day 14 pi. It is anticipated that results from this study may lead to new treatment strategies to attenuate the development of HSK.
31 Type I IFNs enhance metabolic activation to enable an antiviral response Daniel J. Burke a, Leonidas C. Platanias b, Eleanor N. Fish a, a Toronto General Research Institute, University Health Network & Dept. of Immunology, University of Toronto, Canada, b Robert H. Lurie Comprehensive Cancer Center and Division of Hematology– Oncology, Feinberg School of Medicine, Northwestern University, Chicago, United States Type I IFNs induce an antiviral immune response within hours of virus infection, mediated by activation of signaling pathways downstream of the type I IFN receptor. An IFN-induced antiviral response requires both transcriptional activation of genes and the rapid translation of mRNAs into proteins that effect an antiviral response. Both these processes are energy taxing. Cognizant that IFNs activate both the PI3K-Akt/mTOR pathway that is associated with nutrient sensing, and AMPK, associated with stress-induced metabolic activation, we undertook studies to investigate whether IFN-alpha/beta treatment influences metabolic events to enable an antiviral response. Using a panel of mouse embryonic fibroblasts null for Akt1/2, p85ß, AMPKa, TSC2 or 4E-BP1, i.e. with defects in various signaling intermediates along the PI3K/mTOR pathway, we provide evidence for IFN-induced regulation of glucose uptake, mediated by activation of the PI3K/mTOR pathway. Notably, we demonstrate that IFN regulation of glucose uptake is required for an antiviral response to infection with the cardiotropic virus, coxsackievirus B3 (CVB3). In addition, we provide evidence for IFN regulated increases in ATP production. In vivo, we provide evidence that activation of AMPK by the pharmacological agent metformin enhances the antiviral effects of IFN-ß treatment in mice infected with CVB3, lending further support that metabolic activation enhances an IFN-induced antiviral response.
http://dx.doi.org/10.1016/j.cyto.2013.06.034
32 IRF7-dependent, type I IFN (IFN-I) production induces lethal immune-mediated disease in STAT1 KO mice infected with LCMV
http://dx.doi.org/10.1016/j.cyto.2013.06.032 Iain L. Campbell, Wen Li, Markus J. Hofer, School of Molecular Bioscience, University of Sydney, NSW, Australia 30 Anti-IL-37 antibody abrogates the protection in IL-37 transgenic mice to LPSinduced septic shock Ana-Maria Bulau a, Andrea Gruschka a, Rahel Schwaiger a, Charles A. Dinarello b, Philip Bufler a, a Department of Pediatrics, Dr. von Hauner Children’s Hospital, LudwigMaximilians-University, Munich, Germany, b University of Colorado Denver, Aurora, CO, USA IL-37 is a fundamental inhibitor of innate immunity by reducing systemic and local inflammation. For example, mice transgenic for the IL-37 precursor (IL-37tg) are protected against LPS-induced septic shock. We have observed that processing of the IL-37 precursor by caspase-1 is required for its anti-inflammatory function in vitro and that IL-37 is secreted into the extracellular space. In the present study, we intended to neutralize secreted IL-37 in transfected macrophage cells and in IL37tg mice in order to determine the functional role of extracellular IL-37. RAW macrophages expressing IL-37 (RAW-IL-37) and mock transfected cells were preincubated with goat anti-human IL-37-IgG (R&D Systems) or control IgG for 4 h and then stimulated with 100 ng/ml LPS for 18 h. Supernatants were analyzed for IL-6.
STAT1 KO but not WT, STAT2 KO, IRF9 KO or IFNAR KO mice succumb to a lethal CD4+ T cell-mediated disease following systemic infection with lymphocytic choriomeningitis virus (LCMV). In previous studies we showed: (i) LCMV infection induces IRF7 gene expression in a STAT1-independent, but STAT2-dependent manner, and (2) IRF7 is required for IFN-alpha but not IFN-besa production in LCMV infection. Here we examined the role of IRF7 in the lethal host response to LCMV infection in STAT1 KO mice. In contrast to STAT1 KO mice, STAT1/IRF7 double KO mice survived LCMV infection and had a marked reduction in immune pathology affecting key organs including the liver, kidney and lung. LCMV infection in STAT1 KO mice was associated with a significant elevation of a number of cytokines in the serum including IFNakpga, IFN-besa, IFN-calla, IL-5, IL-6 and MCP1 but this response was absent in STAT1/IRF7 KO mice which had a modest increase in MCP-1 only. In addition, in the spleen of LCMV-infected STAT1 KO mice, IFN-besa and IFN-calla mRNAs were also much more highly induced when compared with WT mice, while in STAT1/ IRF7 KO mice IFN-calla mRNA induction was similar to WT animals, there was no induction of IFN-besa mRNA. To elucidate further a possible role of IFN-I in lethal disease, we examined STAT1/IFNAR double KO mice. In contrast to STAT1 KO mice that all succumbed to lethal disease, STAT1/IFNAR KO mice all survived infection with LCMV. In conclusion, lethality resulting from LCMV infection in STAT1 KO mice