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Abstract / Cytokine 70 (2014) 28–79
defense against Acinetobacter baumannii infection, wild-type (WT) and IL-10-deficient mice were infected intranasally with the bacteria. Survival rate, the change of body weight, the level of cytokines and chemokines in lung lysate, and lung histopathology were examined. 73% of IL-10 deficient mice (13/21) were died during experimental period, whereas all WT mice survived. The pulmonary bacterial loads were significantly higher in IL-10-deficient mice, as compared with WT mice at 3 day after infection, although there was no significant difference at 1 day after infection. The level of IL-6, TNF-a, CXCL, CCL2, IFN-c, and IL-1b was higher in lung lysate of IL-10 deficient mice as compared with that of WT mice. Moreover, histological findings showed that lung inflammation was much severe in IL-10 deficient mice. Endogenous IL-10 was essential for optimal phagocytic activity of macrophages, and treatment of recombinant IL-10 enhanced the ability of phagocytosis and bacterial killing in macrophages. Also, intranasal administration of recombinant IL-10 rescued mice from lethality by lung infection of A. baumannii. Our results show that IL-10 plays an important role in the host defense against pulmonary infection of A. baumannii.
http://dx.doi.org/10.1016/j.cyto.2014.07.099
93 Identification of the intracellular location and mechanisms of NOD1-dependent inflammatory responses Aaron Irving 1, Hitomi Mimuro 2, Thomas Kufer 3, Camden Lo 4, Lorinda Turner 5, Belinda Thomas 5, John Bertin 6, Ivo Boneca 7, Chihiro Sasakawa 8, Dana Philpott 9, Richard Ferrero 5, Maria Kaparakis-Liaskos 5, 1 Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria, Australia, 2 Division of Bacteriology, Department of Infectious Diseases Control, International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 3 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany, 4 Monash Micro Imaging, Clayton, Victoria, Australia, 5 Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Clayton, Victoria, Australia, 6 GlaxoSmithKline, Collegeville, USA, 7 Institut Pasteur, Paris, France, 8 Division of Bacterial Infection Biology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 9 Department of Immunology, University of Toronto, Toronto, Canada Aims: Nucleotide oligomerization domain 1 (NOD1) is an intracellular host receptor that senses microbial pathogens by detecting a conserved structure of Gram negative bacterial peptidoglycan (PG). Detection of peptidoglycan by NOD1 ultimately results in a pro-inflammatory cytokine response. However, to date, the intracellular location and the mechanisms whereby NOD1 detects peptidoglycan resulting in the development of pro-inflammatory cytokine responses and autophagy are unknown. Methods: We used peptidoglycan-containing bacterial outer membrane vesicles (PG-OMVs) as a tool to elucidate the intracellular location of NOD1 and the mechanisms of NOD1-dependent responses that result in cytokine production and autophagy. Results: Upon entry into host epithelial cells, PG-OMVs from mucosal pathogens induced NOD1-dependent autophagy and IL-8 responses. Fluorescent labelling of peptidoglycan contained within bacterial OMVs revealed that upon entry into host cells, peptidoglycan migrated to early endosomes where it interacted with NOD1 and the NOD1-adaptor protein RIP-2, facilitating the development of an inflammatory response from this location. We showed that migration of PG-OMVs to early endosomes occurred in a NOD1 dependent manner, identifying a previously unknown role for NOD1 in the intracellular migration of peptidoglycan. Most importantly, using fluorescent lifetime imaging microscopy (FLIM)-fluorescence energy transfer (FRET), we were able to show for the first time the direct interaction between bacterial peptidoglycan and NOD1 within host cells. Finally, we found that the NOD1 adaptor protein RIP-2 is essential for the development of NOD1-dependent autophagy and IL-8 production in response to PG-OMVs [1]. Conclusions: This study reveals for the first time the intracellular location and the early recognition events required for the detection of Gram negative bacterial pathogens by NOD1. Moreover, this study is the first to visualise a direct interaction between bacterial peptidoglycan and NOD1. These findings will significantly expand our limited knowledge of the contribution of NOD1 in Gram negative bacterial pathogenesis, innate immunity and inflammatory disorders.
References [1] Irving A et al.. Cell Host Microbe 2014;15(5):623–35. http://dx.doi.org/10.1016/j.cyto.2014.07.100
94 A viral sensor MDA5 and autoimmune diseases Hiroki Kato, Institute for Virus Research, Kyoto University, Japan MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms and also progressive encephalopathy without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5.These findings provide insight into the association between disorders of the innate immune system and autoimmunity.
http://dx.doi.org/10.1016/j.cyto.2014.07.101
95 Sex-differences in classical monocyte recruitment during inflammation Emma Kay, Ramona S. Scotland, James R. Whiteford, William Harvey Research Institute, QMUL, London, United Kingdom Background: Certain human cardiovascular diseases are subject to gender bias with more prevalence in men. Sexual dimorphisms in murine leukocyte responses during inflammation have previously been described, with males recruiting more neutrophils [1]. This study aimed to determine sex-differences in storage, trafficking and responses of classical monocytes (CMs), in an acute inflammatory model. Materials and methods: Peritonitis was induced by intraperitoneal administration of zymosan (1 mg) to CX3CR1þ=gfp mice. Leukocytes were isolated from blood, peritoneal cavity, bone marrow (BM) and spleen. CMs were identified as CX3CR1+ Gr1+ by flow cytometry. Results: Equal basal circulating, peritoneal and BM CM numbers were observed between the sexes, however males exhibited a greater splenic storage pool. Expression of key chemokine receptors was similar on splenic CMs from both sexes, as was the basal splenic chemokine environment. In response to zymosan, male mice demonstrated 2-fold greater classical monocytosis at 3 h post-administration than females, which translated into greater recruitment to the peritoneum. Reduction in BM cells at 3 h indicated mobilization; this was equal in both sexes. Equivalent spleen CM numbers at 3 h suggest greater outward flux in males than females. Female resident peritoneal macrophages were more numerous however expression of zymosansensing TLR2–TLR6 was equal to males and no sex-differences were seen in the basal or inflamed peritoneal cytokine environments. Conclusions: These data indicate that male mice have greater splenic stores of CMs and furthermore recruit more CMs to the tissue during peritonitis. Sex-differences in classical monocyte trafficking during inflammation may translate to monocyte/macrophage-dependant pathologies more prevalent in males, like atherosclerosis, and may provide a more personalised method of targeting such diseases.
Reference [1] Scotland RS et al.. Blood 2011;118:5918–27. http://dx.doi.org/10.1016/j.cyto.2014.07.102
96 PD-L1hi B regulatory cells are critical for control of the humoral immune response Adnan R. Khan 1,2,3,4,5, Emily Hams 1, Tim Sparwasser 2, Casey T. Weaver 3, Padraic G. Fallon 1,4,5, 1 Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, CO DU, Ireland, 2 Institute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hanover, Germany, 3 Department of Pathology, University of Alabama, AL, United States, 4 National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland, 5 Institute of Molecular Medicine, St. James’s Hospital, Dublin, Ireland
Abstract / Cytokine 70 (2014) 28–79
defense against Acinetobacter baumannii infection, wild-type (WT) and IL-10-deficient mice were infected intranasally with the bacteria. Survival rate, the change of body weight, the level of cytokines and chemokines in lung lysate, and lung histopathology were examined. 73% of IL-10 deficient mice (13/21) were died during experimental period, whereas all WT mice survived. The pulmonary bacterial loads were significantly higher in IL-10-deficient mice, as compared with WT mice at 3 day after infection, although there was no significant difference at 1 day after infection. The level of IL-6, TNF-a, CXCL, CCL2, IFN-c, and IL-1b was higher in lung lysate of IL-10 deficient mice as compared with that of WT mice. Moreover, histological findings showed that lung inflammation was much severe in IL-10 deficient mice. Endogenous IL-10 was essential for optimal phagocytic activity of macrophages, and treatment of recombinant IL-10 enhanced the ability of phagocytosis and bacterial killing in macrophages. Also, intranasal administration of recombinant IL-10 rescued mice from lethality by lung infection of A. baumannii. Our results show that IL-10 plays an important role in the host defense against pulmonary infection of A. baumannii.
http://dx.doi.org/10.1016/j.cyto.2014.07.099
93 Identification of the intracellular location and mechanisms of NOD1-dependent inflammatory responses Aaron Irving 1, Hitomi Mimuro 2, Thomas Kufer 3, Camden Lo 4, Lorinda Turner 5, Belinda Thomas 5, John Bertin 6, Ivo Boneca 7, Chihiro Sasakawa 8, Dana Philpott 9, Richard Ferrero 5, Maria Kaparakis-Liaskos 5, 1 Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria, Australia, 2 Division of Bacteriology, Department of Infectious Diseases Control, International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 3 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany, 4 Monash Micro Imaging, Clayton, Victoria, Australia, 5 Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Clayton, Victoria, Australia, 6 GlaxoSmithKline, Collegeville, USA, 7 Institut Pasteur, Paris, France, 8 Division of Bacterial Infection Biology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan, 9 Department of Immunology, University of Toronto, Toronto, Canada Aims: Nucleotide oligomerization domain 1 (NOD1) is an intracellular host receptor that senses microbial pathogens by detecting a conserved structure of Gram negative bacterial peptidoglycan (PG). Detection of peptidoglycan by NOD1 ultimately results in a pro-inflammatory cytokine response. However, to date, the intracellular location and the mechanisms whereby NOD1 detects peptidoglycan resulting in the development of pro-inflammatory cytokine responses and autophagy are unknown. Methods: We used peptidoglycan-containing bacterial outer membrane vesicles (PG-OMVs) as a tool to elucidate the intracellular location of NOD1 and the mechanisms of NOD1-dependent responses that result in cytokine production and autophagy. Results: Upon entry into host epithelial cells, PG-OMVs from mucosal pathogens induced NOD1-dependent autophagy and IL-8 responses. Fluorescent labelling of peptidoglycan contained within bacterial OMVs revealed that upon entry into host cells, peptidoglycan migrated to early endosomes where it interacted with NOD1 and the NOD1-adaptor protein RIP-2, facilitating the development of an inflammatory response from this location. We showed that migration of PG-OMVs to early endosomes occurred in a NOD1 dependent manner, identifying a previously unknown role for NOD1 in the intracellular migration of peptidoglycan. Most importantly, using fluorescent lifetime imaging microscopy (FLIM)-fluorescence energy transfer (FRET), we were able to show for the first time the direct interaction between bacterial peptidoglycan and NOD1 within host cells. Finally, we found that the NOD1 adaptor protein RIP-2 is essential for the development of NOD1-dependent autophagy and IL-8 production in response to PG-OMVs [1]. Conclusions: This study reveals for the first time the intracellular location and the early recognition events required for the detection of Gram negative bacterial pathogens by NOD1. Moreover, this study is the first to visualise a direct interaction between bacterial peptidoglycan and NOD1. These findings will significantly expand our limited knowledge of the contribution of NOD1 in Gram negative bacterial pathogenesis, innate immunity and inflammatory disorders.
References [1] Irving A et al.. Cell Host Microbe 2014;15(5):623–35. http://dx.doi.org/10.1016/j.cyto.2014.07.100
94 A viral sensor MDA5 and autoimmune diseases Hiroki Kato, Institute for Virus Research, Kyoto University, Japan MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms and also progressive encephalopathy without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5.These findings provide insight into the association between disorders of the innate immune system and autoimmunity.
http://dx.doi.org/10.1016/j.cyto.2014.07.101
95 Sex-differences in classical monocyte recruitment during inflammation Emma Kay, Ramona S. Scotland, James R. Whiteford, William Harvey Research Institute, QMUL, London, United Kingdom Background: Certain human cardiovascular diseases are subject to gender bias with more prevalence in men. Sexual dimorphisms in murine leukocyte responses during inflammation have previously been described, with males recruiting more neutrophils [1]. This study aimed to determine sex-differences in storage, trafficking and responses of classical monocytes (CMs), in an acute inflammatory model. Materials and methods: Peritonitis was induced by intraperitoneal administration of zymosan (1 mg) to CX3CR1þ=gfp mice. Leukocytes were isolated from blood, peritoneal cavity, bone marrow (BM) and spleen. CMs were identified as CX3CR1+ Gr1+ by flow cytometry. Results: Equal basal circulating, peritoneal and BM CM numbers were observed between the sexes, however males exhibited a greater splenic storage pool. Expression of key chemokine receptors was similar on splenic CMs from both sexes, as was the basal splenic chemokine environment. In response to zymosan, male mice demonstrated 2-fold greater classical monocytosis at 3 h post-administration than females, which translated into greater recruitment to the peritoneum. Reduction in BM cells at 3 h indicated mobilization; this was equal in both sexes. Equivalent spleen CM numbers at 3 h suggest greater outward flux in males than females. Female resident peritoneal macrophages were more numerous however expression of zymosansensing TLR2–TLR6 was equal to males and no sex-differences were seen in the basal or inflamed peritoneal cytokine environments. Conclusions: These data indicate that male mice have greater splenic stores of CMs and furthermore recruit more CMs to the tissue during peritonitis. Sex-differences in classical monocyte trafficking during inflammation may translate to monocyte/macrophage-dependant pathologies more prevalent in males, like atherosclerosis, and may provide a more personalised method of targeting such diseases.
Reference [1] Scotland RS et al.. Blood 2011;118:5918–27. http://dx.doi.org/10.1016/j.cyto.2014.07.102
96 PD-L1hi B regulatory cells are critical for control of the humoral immune response Adnan R. Khan 1,2,3,4,5, Emily Hams 1, Tim Sparwasser 2, Casey T. Weaver 3, Padraic G. Fallon 1,4,5, 1 Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, CO DU, Ireland, 2 Institute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hanover, Germany, 3 Department of Pathology, University of Alabama, AL, United States, 4 National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland, 5 Institute of Molecular Medicine, St. James’s Hospital, Dublin, Ireland