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Ficolins promote fungal clearance and modulate the inflammatory cytokine response in host defense against Aspergillus fumigatus
Abstracts / Immunobiology 221 (2016) 1131–1225
14 Excretions/secretions from medicinal larvae (Lucilia sericata) inhibit complement activation by two mechanisms Tetsuro Tamura 1,2,∗ , Gwendolyn Cazander 3 , Suzan H.M. Rooijakkers 4 , Leendert A. Trouw 5 , Peter H. Nibbering 1 1 Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 3 Department of Surgery, Medical Center Haaglanden/Bronovo Hospital, The Hague, The Netherlands 4 Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands 5 Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
Introduction: Larvae of the blowfly Lucilia sericata facilitate wound healing by removing dead tissue and biofilms from nonhealing and necrotic wounds. Another beneficial action of larvae and their excretions/secretions (ES) is down-regulation of excessive inflammation. Aim: As prolonged complement activation is key to excessive inflammation, the aim of this study was to elucidate the mechanisms underlying the anti-complement activities of ES. Results: Our results revealed that heat sensitive serine protease(s) in ES degrade multiple complement proteins in all steps of the three complement activation pathways. Importantly, C3a and C5a – major activators of inflammation – were also degraded by ES, and pretreatment of these factors with ES completely blocked their ability to induce a rise in intracellular free Ca2+ concentration in human neutrophils. Exposure of the neutrophils to ES did not affect their responsiveness to C3a/C5a and fMLP, indicating that the receptors for these activators on neutrophils were not affected by ES. Surprisingly, heat and the serine protease inhibitor pretreatment did not affect the ability of ES to inhibit C5b-9 complex formation, indicating a second complement-inhibiting molecule in ES. Heated ES was as effective as intact ES in inhibiting C3 deposition upon activation of the alternative pathway, but was significantly less effective in wells with a classical or lectin pathway-specific coating. The molecule regulating the C3 amplification loop could not be identified yet. Conclusion: Together, larval ES inhibit complement activation by two different mechanisms and down-regulate the C3a/C5amediated neutrophil activation. This attenuates the inflammatory process, which may facilitate wound healing. http://dx.doi.org/10.1016/j.imbio.2016.06.029 15 The roles of ribosomal protein S19 polymer in acute pleurisy model C57BL/6J mice Hiroshi Nishiura ∗ , Koji Yamanegi, Nahoko Yamada, Yasuko Yagaki, Keiji Nakasho Department of Pathology, Hyogo College of Medicine, Nishinomiya, Japan The macrophage-derived cytokine stream is one of causes of rheumatoid arthritis-synovium. To study a mechanism of predominant macrophage infiltration, we prepared extracts of rheumatoid
1137
arthritis-synovial tissues and found S19 ribosomal protein (RP S19) polymer as monocyte predominate chemotaxis factor. We recently found that apoptotic cells express C5a receptor and produce its antagonist/agonist ligand cross-linked RP S19 between Lys122 and Gln137 by tissue transglutaminases. RP S19 polymer participates in a short lifespan of apoptotic cells via an activation of apoptosis inducing transcription factor delta lactoferrin, which is a neutrophil-specific protein, in an autocrine manner. In addition, RP S19 polymer plays a part in a strong phagocytic clearance of macrophages via an activation of calcium channel opening factor annexin A3, which is a macrophage-specific protein, in a paracrine manner. To validate the roles of RP S19 polymer in vivo, we prepared C57BL/6J mice with Gln137Glu mutant RP S19 gene. Instead of neutrophils, we prepared side population (SP) cells in bone marrows using FACSAriaIII. Lifespan of SP cells in vitro culture condition was postponed by knocked-in mice. We next prepared carrageenan-induced and immune complexinduced acute pleurisy model mice. Cells in lung cavities or lung and spleen parenchyma were collected at 24 h and 7 days after an injection of materials into lung cavities. In contrast to control mice, neutrophil cell numbers in lung cavities of knocked-in mice were large at 24 h. Interestingly, huge numbers of neutrophils were still observed in lung cavities of knocked-in mice at 7 days. Surprisingly, huge numbers of macrophages were also observed in the same samples. In these experimental settings, we observed the neutrophil infiltration in lung parenchyma at 24 h. Moreover, granuloma-like formation was observed in knocked-in mice at 7 days. In addition to lung parenchyma, the weight of spleen parenchyma was increased by knocked-in mice at 24 h. The knocked-in mice-induced extension of acute inflammation was rescued by a supplement of RP S19 polymer functional analogue. These above data indicated the roles of RP S19 polymer in phagocytic clearance of the infiltrating neutrophils by macrophages. We are now interested in a role of programmed cell death in a prevention of shifting from acute inflammation to chronic inflammation. http://dx.doi.org/10.1016/j.imbio.2016.06.030 16 Ficolins promote fungal clearance and modulate the inflammatory cytokine response in host defense against Aspergillus fumigatus Genster Ninette 1,∗ , Cramer Elisabeth Præstekjær 2 , Rosbjerg Anne 1 , Pilely Katrine 1 , Cowland Jack 2 , Garred Peter 1 1 Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 The Granulocyte Research Laboratory, Department of Hematology, Copenhagen University Hospital, Copenhagen, Denmark
Aspergillus fumigatus is an opportunistic fungal pathogen causing severe and usually fatal invasive infections in immunocompromised patients. Innate immunity, including effector cells and pattern recognition receptors, plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus. While a protective effect of ficolins has been demonstrated in mouse models of bacterial and viral infections, their role in host defense against fungal infections in vivo
1138
Abstracts / Immunobiology 221 (2016) 1131–1225
is unknown. In this study we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 hours post-infection compared to wildtype mice. The delayed clearance of A. fumigatus conidia from the lungs of ficolin knockout mice could not be attributed to compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited decreased expression and protein levels of pro-inflammatory cytokines in the lungs compared to wildtype mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement-activation in ficolin knockout mice and wildtype mice. Taken together these results suggest that ficolins promote fungal clearance of A. fumigatus and modulate the inflammatory response independent of complement activation. In conclusion this study demonstrates that ficolins are important in the initial innate host defense against A. fumigatus infections in vivo. http://dx.doi.org/10.1016/j.imbio.2016.06.031 17 Cholesterol crystals activate the lectin complement pathway via ficolin-2 and MBL – Implications for the progression of atherosclerosis Katrine Pilely 1,∗ , Anne Rosbjerg 1 , Ninette Genster 1 , Peter Gal 2 , Gábor Pál 3 , Bente Halvorsen 4,5,6 , Sverre Holm 4,7 , Pål Aukrust 4,5,6 , Siril Skaret Bakke 8 , Bjørnar Sporsheim 8 , Ingunn Nervik 9 , Nathalie Niyonzima 8 , Emil D. Bartels 10 , Gregory L. Stahl 11 , Tom Eirik Mollnes 12,13,8 , Terje Espevik 8 , Peter Garred 1 1 Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary 3 Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary 4 Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway 5 Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway 6 K.G. Jebsen Inflammation Research Center, University of Oslo, Oslo, Norway 7 Hospital for Rheumatic Diseases, Lillehammer, Norway 8 Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway 9 Department of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology, Trondheim, Norway 10 Department of Clinical Biochemistry, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 11 Center for Experimental Therapeutics and Reperfusion Injury, Harvard Institutes of Medicine, Boston, MA, USA
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Department of Immunology, Oslo University Hospital, K.G. Jebsen IRC, University of Oslo, Norway 13 Research Laboratory, Nordland Hospital, Bodø, K.G. Jebsen TREC, University of Tromsø, Norway Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRM) from the lectin pathway bind CC and functions as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors as well as deficient and normal sera. In addition we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium dependent manner while ficolin-2 binding was calcium independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis. http://dx.doi.org/10.1016/j.imbio.2016.06.032 18 Regulation of TLR9-mediated human B cell functions by C3a and C3adesArg – A novel anti-inflammatory effect of C3a on the humoral immune response Mariann Kremlitzka 1,∗ , Zsófia Csáti 2 , Christian M. Karsten 3 , Jörg Köhl 3,4 , Anna Erdei 1,2 1
MTA-ELTE Immunology Research Group, Eötvös Loránd University, Budapest, Hungary 2 Eötvös Loránd University, Department of Immunology, Budapest, Hungary 3 University of Lübeck, Institute for Systemic Inflammation Research, Lübeck, Germany 4 Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Background: The complement system and Toll-like receptors (TLRs) are two essential parts of innate immunity that provide first line of defense against invading pathogens. The regulation of the humoral immune response by larger fragments of C3, i.e. C3b and C3d/g, and TLRs is well appreciated. In contrast, the effect of C3a and its interaction with TLRs on human B cell responses are poorly defined. In this study, we assessed the expression of the C3a receptor (C3aR) in human B cells and the effects of C3a and C3a(desArg) on TLR9-induced B cell functions. Methods: Human B cells were separated from peripheral blood of healthy donors and C3aR expression was determined at the mRNA level by reverse transcription PCR and at the protein level by Western Blot, flow cytometry and confocal microscopy. Isolated B cells were stimulated via TLR9 using the synthetic activator
14 Excretions/secretions from medicinal larvae (Lucilia sericata) inhibit complement activation by two mechanisms Tetsuro Tamura 1,2,∗ , Gwendolyn Cazander 3 , Suzan H.M. Rooijakkers 4 , Leendert A. Trouw 5 , Peter H. Nibbering 1 1 Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 3 Department of Surgery, Medical Center Haaglanden/Bronovo Hospital, The Hague, The Netherlands 4 Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands 5 Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
Introduction: Larvae of the blowfly Lucilia sericata facilitate wound healing by removing dead tissue and biofilms from nonhealing and necrotic wounds. Another beneficial action of larvae and their excretions/secretions (ES) is down-regulation of excessive inflammation. Aim: As prolonged complement activation is key to excessive inflammation, the aim of this study was to elucidate the mechanisms underlying the anti-complement activities of ES. Results: Our results revealed that heat sensitive serine protease(s) in ES degrade multiple complement proteins in all steps of the three complement activation pathways. Importantly, C3a and C5a – major activators of inflammation – were also degraded by ES, and pretreatment of these factors with ES completely blocked their ability to induce a rise in intracellular free Ca2+ concentration in human neutrophils. Exposure of the neutrophils to ES did not affect their responsiveness to C3a/C5a and fMLP, indicating that the receptors for these activators on neutrophils were not affected by ES. Surprisingly, heat and the serine protease inhibitor pretreatment did not affect the ability of ES to inhibit C5b-9 complex formation, indicating a second complement-inhibiting molecule in ES. Heated ES was as effective as intact ES in inhibiting C3 deposition upon activation of the alternative pathway, but was significantly less effective in wells with a classical or lectin pathway-specific coating. The molecule regulating the C3 amplification loop could not be identified yet. Conclusion: Together, larval ES inhibit complement activation by two different mechanisms and down-regulate the C3a/C5amediated neutrophil activation. This attenuates the inflammatory process, which may facilitate wound healing. http://dx.doi.org/10.1016/j.imbio.2016.06.029 15 The roles of ribosomal protein S19 polymer in acute pleurisy model C57BL/6J mice Hiroshi Nishiura ∗ , Koji Yamanegi, Nahoko Yamada, Yasuko Yagaki, Keiji Nakasho Department of Pathology, Hyogo College of Medicine, Nishinomiya, Japan The macrophage-derived cytokine stream is one of causes of rheumatoid arthritis-synovium. To study a mechanism of predominant macrophage infiltration, we prepared extracts of rheumatoid
1137
arthritis-synovial tissues and found S19 ribosomal protein (RP S19) polymer as monocyte predominate chemotaxis factor. We recently found that apoptotic cells express C5a receptor and produce its antagonist/agonist ligand cross-linked RP S19 between Lys122 and Gln137 by tissue transglutaminases. RP S19 polymer participates in a short lifespan of apoptotic cells via an activation of apoptosis inducing transcription factor delta lactoferrin, which is a neutrophil-specific protein, in an autocrine manner. In addition, RP S19 polymer plays a part in a strong phagocytic clearance of macrophages via an activation of calcium channel opening factor annexin A3, which is a macrophage-specific protein, in a paracrine manner. To validate the roles of RP S19 polymer in vivo, we prepared C57BL/6J mice with Gln137Glu mutant RP S19 gene. Instead of neutrophils, we prepared side population (SP) cells in bone marrows using FACSAriaIII. Lifespan of SP cells in vitro culture condition was postponed by knocked-in mice. We next prepared carrageenan-induced and immune complexinduced acute pleurisy model mice. Cells in lung cavities or lung and spleen parenchyma were collected at 24 h and 7 days after an injection of materials into lung cavities. In contrast to control mice, neutrophil cell numbers in lung cavities of knocked-in mice were large at 24 h. Interestingly, huge numbers of neutrophils were still observed in lung cavities of knocked-in mice at 7 days. Surprisingly, huge numbers of macrophages were also observed in the same samples. In these experimental settings, we observed the neutrophil infiltration in lung parenchyma at 24 h. Moreover, granuloma-like formation was observed in knocked-in mice at 7 days. In addition to lung parenchyma, the weight of spleen parenchyma was increased by knocked-in mice at 24 h. The knocked-in mice-induced extension of acute inflammation was rescued by a supplement of RP S19 polymer functional analogue. These above data indicated the roles of RP S19 polymer in phagocytic clearance of the infiltrating neutrophils by macrophages. We are now interested in a role of programmed cell death in a prevention of shifting from acute inflammation to chronic inflammation. http://dx.doi.org/10.1016/j.imbio.2016.06.030 16 Ficolins promote fungal clearance and modulate the inflammatory cytokine response in host defense against Aspergillus fumigatus Genster Ninette 1,∗ , Cramer Elisabeth Præstekjær 2 , Rosbjerg Anne 1 , Pilely Katrine 1 , Cowland Jack 2 , Garred Peter 1 1 Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 The Granulocyte Research Laboratory, Department of Hematology, Copenhagen University Hospital, Copenhagen, Denmark
Aspergillus fumigatus is an opportunistic fungal pathogen causing severe and usually fatal invasive infections in immunocompromised patients. Innate immunity, including effector cells and pattern recognition receptors, plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus. While a protective effect of ficolins has been demonstrated in mouse models of bacterial and viral infections, their role in host defense against fungal infections in vivo
1138
Abstracts / Immunobiology 221 (2016) 1131–1225
is unknown. In this study we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 hours post-infection compared to wildtype mice. The delayed clearance of A. fumigatus conidia from the lungs of ficolin knockout mice could not be attributed to compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited decreased expression and protein levels of pro-inflammatory cytokines in the lungs compared to wildtype mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement-activation in ficolin knockout mice and wildtype mice. Taken together these results suggest that ficolins promote fungal clearance of A. fumigatus and modulate the inflammatory response independent of complement activation. In conclusion this study demonstrates that ficolins are important in the initial innate host defense against A. fumigatus infections in vivo. http://dx.doi.org/10.1016/j.imbio.2016.06.031 17 Cholesterol crystals activate the lectin complement pathway via ficolin-2 and MBL – Implications for the progression of atherosclerosis Katrine Pilely 1,∗ , Anne Rosbjerg 1 , Ninette Genster 1 , Peter Gal 2 , Gábor Pál 3 , Bente Halvorsen 4,5,6 , Sverre Holm 4,7 , Pål Aukrust 4,5,6 , Siril Skaret Bakke 8 , Bjørnar Sporsheim 8 , Ingunn Nervik 9 , Nathalie Niyonzima 8 , Emil D. Bartels 10 , Gregory L. Stahl 11 , Tom Eirik Mollnes 12,13,8 , Terje Espevik 8 , Peter Garred 1 1 Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary 3 Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary 4 Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway 5 Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway 6 K.G. Jebsen Inflammation Research Center, University of Oslo, Oslo, Norway 7 Hospital for Rheumatic Diseases, Lillehammer, Norway 8 Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway 9 Department of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology, Trondheim, Norway 10 Department of Clinical Biochemistry, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 11 Center for Experimental Therapeutics and Reperfusion Injury, Harvard Institutes of Medicine, Boston, MA, USA
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Department of Immunology, Oslo University Hospital, K.G. Jebsen IRC, University of Oslo, Norway 13 Research Laboratory, Nordland Hospital, Bodø, K.G. Jebsen TREC, University of Tromsø, Norway Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRM) from the lectin pathway bind CC and functions as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors as well as deficient and normal sera. In addition we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium dependent manner while ficolin-2 binding was calcium independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis. http://dx.doi.org/10.1016/j.imbio.2016.06.032 18 Regulation of TLR9-mediated human B cell functions by C3a and C3adesArg – A novel anti-inflammatory effect of C3a on the humoral immune response Mariann Kremlitzka 1,∗ , Zsófia Csáti 2 , Christian M. Karsten 3 , Jörg Köhl 3,4 , Anna Erdei 1,2 1
MTA-ELTE Immunology Research Group, Eötvös Loránd University, Budapest, Hungary 2 Eötvös Loránd University, Department of Immunology, Budapest, Hungary 3 University of Lübeck, Institute for Systemic Inflammation Research, Lübeck, Germany 4 Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA Background: The complement system and Toll-like receptors (TLRs) are two essential parts of innate immunity that provide first line of defense against invading pathogens. The regulation of the humoral immune response by larger fragments of C3, i.e. C3b and C3d/g, and TLRs is well appreciated. In contrast, the effect of C3a and its interaction with TLRs on human B cell responses are poorly defined. In this study, we assessed the expression of the C3a receptor (C3aR) in human B cells and the effects of C3a and C3a(desArg) on TLR9-induced B cell functions. Methods: Human B cells were separated from peripheral blood of healthy donors and C3aR expression was determined at the mRNA level by reverse transcription PCR and at the protein level by Western Blot, flow cytometry and confocal microscopy. Isolated B cells were stimulated via TLR9 using the synthetic activator