- Email: [email protected]
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Abstract / Cytokine 63 (2013) 243–314 186 Role of GTP-mediated structural changes of MxA on its antiviral function against influenza A
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severity of inflammation by exploiting the inhibitory properties of SIGIRR and by associating with IL-18Ra. http://dx.doi.org/10.1016/j.cyto.2013.06.190
Patricia E. Nigg, Dominik Müller, Jovan Pavlovic, Institute of Medical Virology, University of Zurich, Zurich, Switzerland Mx proteins are interferon type I induced effector proteins and members of the family of dynamin-like GTPases. Human MxA protein exerts antiviral activity against a broad range of viruses including influenza A. The GTPase activity of MxA is essential for its proper antiviral function. Based on extensive mutational biochemical analyses we proposed a model where Mx proteins can assemble into highly ordered oligomers but exert their antiviral activity as monomers. Moreover, we have shown that human MxA physically interacts with the cellular DExD/h-box RNA helicases UAP56 and URH49 that are required for efficient replication of influenza A virus. Recently Gao and coworkers determined the crystal structure of MxA, revealing a globular GTPbinding domain and a four helical bundle stalk domain. In vitro, MxA multimerizes into large ring-like structures, involving three interaction interfaces. Mutations in the interfaces of MxA interfere with the oligomer formation, but, depending on the mutation, some MxA variants are still able to form monomers, dimers or tetramers. To date it is still a matter of debate whether MxA exerts antiviral activity in form of monomers, dimers and/or higher oligomeric structures. In order to assess whether GTP-binding has an effect on the stoichiometry of MxA we purified recombinant wildtype MxA and various mutants thereof and assessed the number of MxA protomers in the presence or absence GTP-cS by multi angle laser light scattering (MALLS). Surprisingly, we observed that in the presence of GTP-cS wildtype MxA converted from oligomers to tetramers and dimers. Similarly, addition of GTP-cS to the tetrameric mutant MxA (R640A) resulted in its dimerization. Furthermore, dimeric and monomeric MxA variants did not alter their stoichiometry upon addition of GTP-cS. Further, we evaluated the antiviral activity of wild type MxA and MxA variants employing an influenza A minireplicon system and influenza A virus infection of MxA expressing cells. The data clearly indicated that certain tetrameric, dimeric or monomeric variants of MxA were still able to efficiently inhibit virus replication while MxA mutants deficient for GTP binding were inactive. We currently test whether binding of UAP56 by MxA is required for its antiviral activity.
http://dx.doi.org/10.1016/j.cyto.2013.06.189
187 Interleukin 37 employs the IL-1 family inhibitory receptor SIGIRR and the alpha chain of the IL-18 receptor to suppress innate immunity Marcel F. Nold a,b, Claudia A. Nold-Petry a,b, Camden Lo c, Suzhao Li b, Ina Rudloff a, Jarod A. Zepp b, Tania Azam b, Philip Bufler d, Cecilia Garlanda e, Alberto Mantovani e, Charles A. Dinarello b, a The Ritchie Centre, Monash Institute of Medical Research, Melbourne, Victoria, Australia, b Department of Medicine, University of Colorado Denver, USA, c Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia, d Children’s Hospital, Ludwig-Maximilians University, Munich, Germany, e Istituto Clinico Humanitas, Milan, Italy IL-1 family member IL-37 inhibits a broad spectrum of inflammatory assaults in cell lines and primary PBMC; in vivo, mice transgenic for IL-37 (IL-37tg) exhibit markedly reduced manifestations of LPS-induced shock, DSS colitis, antigen-specific immune responses and ischemia–reperfusion injury. Although previous studies reported that recombinant IL-37 associates with the IL-18 receptor alpha chain (IL-18Ra), no biological activity was observed. Here, we reveal a functional role of IL-18Ra for the anti-inflammatory properties of IL-37. We also identify that the inhibitory IL-1R family member, SIGIRR, associates with IL-37 and is required by IL37tg mice for protection from LPS-induced shock. In IL-37-transfected THP-1 macrophages, we observed an 83% reduction in IL-1b, but only a 34% reduction when endogenous SIGIRR was silenced. A similar attenuation of the anti-inflammatory effects was demonstrated in LPS-stimulated human PBMC and by silencing of IL-18Ra. By immunofluorescence, we observed that IL-37 associates with SIGIRR and IL-18Ra in LPSstimulated RAW macrophages and in IL-1b-treated A549 cells, both transfected with IL-37. Using proximity ligation assays and FRET in PBMC, thus exploring interactions of the naturally occurring pairs IL-37-SIGIRR, IL-37-IL-18Ra and SIGIRR-IL-18Ra, we demonstrated sub-40 nm co-localization of each complex. The interactions were hardly detectable at steady-state, reached maximal association 15 min after LPS stimulation, and decreased to background by 6 h. We next generated a strain of SIGIRR-KO mice that are also transgenic for IL-37 (IL-37tg-SIGIRR-KO). As expected, the systemic response to LPS was markedly reduced in IL-37tg mice; however, there was no reduction in IL-37tg-SIGIRR-KO mice. For example, LPS induced severe hypothermia (trough at 25 °C) and acidosis (pH 7.16) in wild-type mice; however, disease severity was markedly ameliorated in IL-37tg mice (29 °C, pH 7.32). In IL-37tg-SIGIRR-KO mice, this protection was considerably weaker. We conclude that IL-37 limits the
188 Dengue infection generates an early NADPH-oxidase dependent ROS production required for antiviral and inflammatory responses David Olagnier, Carmen Nichols, Zhong He, John Hiscott, Vaccine & Gene Therapy Institute of Florida, Port Saint Lucie, FL, United States The rapid host response to virus infection is crucial for establishment of the antiviral state and for the release of pro-inflammatory mediators that coordinate the adaptive immune response. Induction of antiviral and inflammatory genes is achieved largely through recognition of viral nucleic acid motifs by pattern recognition receptors. Dengue virus (DENV) is a significant human pathogen that causes a wide spectrum of clinical symptoms, ranging from mild dengue fever to severe hemorrhagic fever and shock syndrome. The cytosolic receptors RIG-I/MDA5 as well as the membrane-bound TLR3/TLR7 localized in the endosomes are important sensors of DENV infection. The role of reactive oxygen species (ROS) in the activation of biological processes such as autophagy, necrosis, and inflammasome activation adds another layer of complexity to the regulation of immune sensing. Transcriptome analysis of DENVinfected human monocyte-derived dendritic cells (Mo-DC) revealed the activation of distinct patterns of gene transcription, with involvement of pathways including the antiviral response, the TNF superfamily and late activation of death receptor signaling. Interestingly, a dominant early gene network associated with the Nrf2-dependent antioxidant response was also identified; DENV infection induced an early production of ROS that was dependent on NADPH-oxidase, and was essential for the activation of inflammatory and antiviral programs, through NF-jB and IRF-3 transcription factors, respectively. Using a high throughput qPCR assay, we demonstrated that interfering with NADPH-oxidase inhibited DENV-induced antiviral and inflammatory responses. We also demonstrated that the antioxidant pathway controlled by the transcription factor Nrf2 is a pivotal upstream link to the regulation of DENV-induced antiviral and inflammatory responses under oxidative conditions. In conclusion, these findings demonstrate that ROS production is crucial for effective activation of pathways leading to innate immune responses against DENV infection in human Mo-DC. http://dx.doi.org/10.1016/j.cyto.2013.06.191
189 Differences in cytokine responses during infection with high and low pathogenic Ebola viruses Judith Olejnik a,b, Andrea Marzi c, Heinz Feldmann c, Elke Mühlberger a,b, a Department of Microbiology, Boston University, Boston, MA, United States, b National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA, United States, c Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States To date five Ebolavirus species have been identified, differing in pathogenicity. The highly pathogenic Zaire ebolavirus (EBOV) causes a hemorrhagic disease in humans with case fatality rates up to 90%. A hallmark of EBOV infection is a dysregulated immune response, including high levels of cytokines. In contrast, Reston ebolavirus (RESTV) seems to be nonpathogenic in humans, as only asymptomatic cases of RESTV infection in humans have been reported. The mechanisms of protection against RESTV in humans are not yet understood. As cytokines might play a role in the pathogenesis of EBOV infection, we analyzed the cytokine response in EBOV- and RESTVinfected human macrophages and dendritic cells. These cells not only orchestrate innate and adaptive immune responses but are also early targets of Ebolaviruses. We observed degradation of IjBa and nuclear translocation of p65 in EBOV-infected macrophages, indicating activation of NFjB signaling. qRT-PCR and Luminex assays revealed induction of selected cytokines in EBOV-infected macrophages. Most analyzed cytokines and chemokines were upregulated in EBOV infection at 6 hpi or later, whereas others, including IL10, did not show increased secretion levels. In contrast, p65 was not translocated into the nucleus in RESTV-infected macrophages and no significant changes in cytokine mRNA levels or secreted protein levels were observed. To investigate if this effect is due to inhibition of pro-inflammatory signaling pathways, RESTV-infected macrophages were treated with LPS at 1 dpi and cytokine production was determined. Similar levels of pro-inflammatory cytokines, including IL6 and TNFa, were detected in RESTV-infected and LPS-treated macrophages compared to non-infected LPS-treated cells, suggesting that RESTV is not able to inhibit LPS-mediated cytokine induction. Our data support the hypothesis that high pathogenicity
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severity of inflammation by exploiting the inhibitory properties of SIGIRR and by associating with IL-18Ra. http://dx.doi.org/10.1016/j.cyto.2013.06.190
Patricia E. Nigg, Dominik Müller, Jovan Pavlovic, Institute of Medical Virology, University of Zurich, Zurich, Switzerland Mx proteins are interferon type I induced effector proteins and members of the family of dynamin-like GTPases. Human MxA protein exerts antiviral activity against a broad range of viruses including influenza A. The GTPase activity of MxA is essential for its proper antiviral function. Based on extensive mutational biochemical analyses we proposed a model where Mx proteins can assemble into highly ordered oligomers but exert their antiviral activity as monomers. Moreover, we have shown that human MxA physically interacts with the cellular DExD/h-box RNA helicases UAP56 and URH49 that are required for efficient replication of influenza A virus. Recently Gao and coworkers determined the crystal structure of MxA, revealing a globular GTPbinding domain and a four helical bundle stalk domain. In vitro, MxA multimerizes into large ring-like structures, involving three interaction interfaces. Mutations in the interfaces of MxA interfere with the oligomer formation, but, depending on the mutation, some MxA variants are still able to form monomers, dimers or tetramers. To date it is still a matter of debate whether MxA exerts antiviral activity in form of monomers, dimers and/or higher oligomeric structures. In order to assess whether GTP-binding has an effect on the stoichiometry of MxA we purified recombinant wildtype MxA and various mutants thereof and assessed the number of MxA protomers in the presence or absence GTP-cS by multi angle laser light scattering (MALLS). Surprisingly, we observed that in the presence of GTP-cS wildtype MxA converted from oligomers to tetramers and dimers. Similarly, addition of GTP-cS to the tetrameric mutant MxA (R640A) resulted in its dimerization. Furthermore, dimeric and monomeric MxA variants did not alter their stoichiometry upon addition of GTP-cS. Further, we evaluated the antiviral activity of wild type MxA and MxA variants employing an influenza A minireplicon system and influenza A virus infection of MxA expressing cells. The data clearly indicated that certain tetrameric, dimeric or monomeric variants of MxA were still able to efficiently inhibit virus replication while MxA mutants deficient for GTP binding were inactive. We currently test whether binding of UAP56 by MxA is required for its antiviral activity.
http://dx.doi.org/10.1016/j.cyto.2013.06.189
187 Interleukin 37 employs the IL-1 family inhibitory receptor SIGIRR and the alpha chain of the IL-18 receptor to suppress innate immunity Marcel F. Nold a,b, Claudia A. Nold-Petry a,b, Camden Lo c, Suzhao Li b, Ina Rudloff a, Jarod A. Zepp b, Tania Azam b, Philip Bufler d, Cecilia Garlanda e, Alberto Mantovani e, Charles A. Dinarello b, a The Ritchie Centre, Monash Institute of Medical Research, Melbourne, Victoria, Australia, b Department of Medicine, University of Colorado Denver, USA, c Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia, d Children’s Hospital, Ludwig-Maximilians University, Munich, Germany, e Istituto Clinico Humanitas, Milan, Italy IL-1 family member IL-37 inhibits a broad spectrum of inflammatory assaults in cell lines and primary PBMC; in vivo, mice transgenic for IL-37 (IL-37tg) exhibit markedly reduced manifestations of LPS-induced shock, DSS colitis, antigen-specific immune responses and ischemia–reperfusion injury. Although previous studies reported that recombinant IL-37 associates with the IL-18 receptor alpha chain (IL-18Ra), no biological activity was observed. Here, we reveal a functional role of IL-18Ra for the anti-inflammatory properties of IL-37. We also identify that the inhibitory IL-1R family member, SIGIRR, associates with IL-37 and is required by IL37tg mice for protection from LPS-induced shock. In IL-37-transfected THP-1 macrophages, we observed an 83% reduction in IL-1b, but only a 34% reduction when endogenous SIGIRR was silenced. A similar attenuation of the anti-inflammatory effects was demonstrated in LPS-stimulated human PBMC and by silencing of IL-18Ra. By immunofluorescence, we observed that IL-37 associates with SIGIRR and IL-18Ra in LPSstimulated RAW macrophages and in IL-1b-treated A549 cells, both transfected with IL-37. Using proximity ligation assays and FRET in PBMC, thus exploring interactions of the naturally occurring pairs IL-37-SIGIRR, IL-37-IL-18Ra and SIGIRR-IL-18Ra, we demonstrated sub-40 nm co-localization of each complex. The interactions were hardly detectable at steady-state, reached maximal association 15 min after LPS stimulation, and decreased to background by 6 h. We next generated a strain of SIGIRR-KO mice that are also transgenic for IL-37 (IL-37tg-SIGIRR-KO). As expected, the systemic response to LPS was markedly reduced in IL-37tg mice; however, there was no reduction in IL-37tg-SIGIRR-KO mice. For example, LPS induced severe hypothermia (trough at 25 °C) and acidosis (pH 7.16) in wild-type mice; however, disease severity was markedly ameliorated in IL-37tg mice (29 °C, pH 7.32). In IL-37tg-SIGIRR-KO mice, this protection was considerably weaker. We conclude that IL-37 limits the
188 Dengue infection generates an early NADPH-oxidase dependent ROS production required for antiviral and inflammatory responses David Olagnier, Carmen Nichols, Zhong He, John Hiscott, Vaccine & Gene Therapy Institute of Florida, Port Saint Lucie, FL, United States The rapid host response to virus infection is crucial for establishment of the antiviral state and for the release of pro-inflammatory mediators that coordinate the adaptive immune response. Induction of antiviral and inflammatory genes is achieved largely through recognition of viral nucleic acid motifs by pattern recognition receptors. Dengue virus (DENV) is a significant human pathogen that causes a wide spectrum of clinical symptoms, ranging from mild dengue fever to severe hemorrhagic fever and shock syndrome. The cytosolic receptors RIG-I/MDA5 as well as the membrane-bound TLR3/TLR7 localized in the endosomes are important sensors of DENV infection. The role of reactive oxygen species (ROS) in the activation of biological processes such as autophagy, necrosis, and inflammasome activation adds another layer of complexity to the regulation of immune sensing. Transcriptome analysis of DENVinfected human monocyte-derived dendritic cells (Mo-DC) revealed the activation of distinct patterns of gene transcription, with involvement of pathways including the antiviral response, the TNF superfamily and late activation of death receptor signaling. Interestingly, a dominant early gene network associated with the Nrf2-dependent antioxidant response was also identified; DENV infection induced an early production of ROS that was dependent on NADPH-oxidase, and was essential for the activation of inflammatory and antiviral programs, through NF-jB and IRF-3 transcription factors, respectively. Using a high throughput qPCR assay, we demonstrated that interfering with NADPH-oxidase inhibited DENV-induced antiviral and inflammatory responses. We also demonstrated that the antioxidant pathway controlled by the transcription factor Nrf2 is a pivotal upstream link to the regulation of DENV-induced antiviral and inflammatory responses under oxidative conditions. In conclusion, these findings demonstrate that ROS production is crucial for effective activation of pathways leading to innate immune responses against DENV infection in human Mo-DC. http://dx.doi.org/10.1016/j.cyto.2013.06.191
189 Differences in cytokine responses during infection with high and low pathogenic Ebola viruses Judith Olejnik a,b, Andrea Marzi c, Heinz Feldmann c, Elke Mühlberger a,b, a Department of Microbiology, Boston University, Boston, MA, United States, b National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA, United States, c Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States To date five Ebolavirus species have been identified, differing in pathogenicity. The highly pathogenic Zaire ebolavirus (EBOV) causes a hemorrhagic disease in humans with case fatality rates up to 90%. A hallmark of EBOV infection is a dysregulated immune response, including high levels of cytokines. In contrast, Reston ebolavirus (RESTV) seems to be nonpathogenic in humans, as only asymptomatic cases of RESTV infection in humans have been reported. The mechanisms of protection against RESTV in humans are not yet understood. As cytokines might play a role in the pathogenesis of EBOV infection, we analyzed the cytokine response in EBOV- and RESTVinfected human macrophages and dendritic cells. These cells not only orchestrate innate and adaptive immune responses but are also early targets of Ebolaviruses. We observed degradation of IjBa and nuclear translocation of p65 in EBOV-infected macrophages, indicating activation of NFjB signaling. qRT-PCR and Luminex assays revealed induction of selected cytokines in EBOV-infected macrophages. Most analyzed cytokines and chemokines were upregulated in EBOV infection at 6 hpi or later, whereas others, including IL10, did not show increased secretion levels. In contrast, p65 was not translocated into the nucleus in RESTV-infected macrophages and no significant changes in cytokine mRNA levels or secreted protein levels were observed. To investigate if this effect is due to inhibition of pro-inflammatory signaling pathways, RESTV-infected macrophages were treated with LPS at 1 dpi and cytokine production was determined. Similar levels of pro-inflammatory cytokines, including IL6 and TNFa, were detected in RESTV-infected and LPS-treated macrophages compared to non-infected LPS-treated cells, suggesting that RESTV is not able to inhibit LPS-mediated cytokine induction. Our data support the hypothesis that high pathogenicity