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C5aR regulates intestinal microbiota composition and controls the induction of gastrointestinal allergic hypersensitivity
Abstracts / Immunobiology 217 (2012) 1129–1222
51 C5aR regulates intestinal microbiota composition and controls the induction of gastrointestinal allergic hypersensitivity Anna T. Reinicke 1 , Anne Herrmann 1 , Florian Baer 2 , Christian Sina 2 , Girish Srinivas 3,4 , Sven Kuenzel 3 , John Baines 3,5 , Jörg Köhl 1,6 1 Institute for Systemic Inflammation Research, University of Luebeck, 23538 Luebeck, Germany 2 Institute of Anatomy, University Hospital Schleswig-Holstein, Lübeck, Germany 3 Max Planck Institute for Evolutionary Biology, Ploen, Germany 4 Department of Dermatology, University of Luebeck, Lübeck, Germany 5 Institute for Experimental Medicine, University of Kiel, Kiel, Germany 6 Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital and University of Cincinnati, College of Medicine, Cincinnati, OH, USA
Allergic immunity provides important protection in host defense against non-infectious environmental toxins and irritants. However, when excessive, this defense may result in allergic disease. Complement C5a is known to control allergic responses in the lung but little is known about its role in allergic responses in the intestine. Here, we adopted a murine model of gastrointestinal allergy in which repeated oral challenge of ovalbumin (OVA) in OVA/alum-sensitized mice induced acute diarrhea. We observed that C5a receptor (C5aR) is required to mount an acute allergic response against oral allergen. Mechanistically, mast cell degranulation is a critical effector in mediating allergic diarrhea. We found that C5aR is crucial for the recruitment and activation of intestinal mucosal mast cells during gastrointestinal allergy by chloroacetate esterase staining of intestine paraffin sections and measurement of the release of the beta-chymase, MCPT-1. Furthermore, C5aR was found to be required for the production of allergen-specific immunoglobulins during allergy that are essential for mast cell-mediated degranulation. Genotype driven changes to gastrointestinal microbiota have been linked to diseases including inflammatory bowel disease (IBD) and allergic diseases. In this context, we examined the composition of bacterial microbiota in jejunum, ilium, caecum and colon of wild type and C5aR deficient littermates using 454 pyrosequencing. Our results show that the intestinal microbiota is significantly altered by C5aR whereby C5aR deficiency resulted in lower diversity of bacterial communities. Further, we observed a decreased frequency of Lactobacillus spp., which has been associated with protective immunity. We conclude that C5aR is an important determinant shaping the composition of the intestinal microbiota and influencing the ability to mount an allergic response in the intestine. http://dx.doi.org/10.1016/j.imbio.2012.08.052 52 C5aR-dependent cell activation by physiological concentrations of C5a-desArg: Insights from a novel label-free cellular assay Edimara S. Reis 1 , Hui Chen 1 , Georgia Sfyroera 2 , Peter N. Monk 3 , Jörg Köhl 4 , Daniel Ricklin 1 , John D. Lambris 1 1
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 National Center for Scientific Research “Democritos”, Athens, Greece 3 University of Sheffield, Sheffield, UK 4 University of Lübeck, Lübeck, Germany The complement anaphylatoxins C3a, C5a, and C5adesArg play critical roles in the induction of inflammation and the modulation
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of innate and acquired immune responses after binding to their G protein-coupled receptors, C3aR and C5aR. The role of C5adesArg in inducing cell activation has been often neglected, since the affinity of C5adesArg for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5adesArg and also C3a, C3adesArg and C5a to induce activation of transfected and primary immune cells. As expected, C3a and C5a, but not C3adesArg were able to induce cell activation. More importantly, our data indicate that physiological concentrations of C5adesArg can induce significant levels of cell activation that are even higher than that achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, since it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of Galpha-i , Galpha-s and Gbeta/gamma proteins upon C5aR engagement by C5a or C5adesArg . Further, mass spectrometric characterization of plasma-derived C5a and C5adesArg provided important insight into the post-translational modification pattern of these anaphylatoxins, which includes glycosylation at Asn64 and partial cysteinylation at Cys27. While the context-specific physiological contribution of C5adesArg has to be further explored, our data suggest that C5adesArg acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status. http://dx.doi.org/10.1016/j.imbio.2012.08.053 53 C5aR-dependent subversion of neutrophils and dysbiotic inflammation George Hajishengallis 1 , Jennifer L. Krauss 2 , Ravi Jotwani 2 , Toshiharu Abe 1 , Martha Triantafilou 3 , Kathy Triantafilou 3 , Robert A. DeAngelis 4 , John D. Lambris 4 1
University of Pennsylvania School of Dental Medicine, Department of Microbiology, Philadelphia, PA, USA 2 University of Louisville, Oral Health and Systemic Disease, Louisville, KY, USA 3 University of Cardiff, Institute of Infection & Immunity, Cardiff, UK 4 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Porphyromonas gingivalis (Pg) was recently shown to remodel the oral microbiota to a dysbiotic state that causes inflammatory periodontitis. Pg-induced dysbiosis requires a functional C5a receptor (C5aR), and the uncontrolled growth of the microbiota suggests a breakdown of immune surveillance in the periodontal tissue. Because >95% of the leukocytes recruited to the tooth-associated biofilm are neutrophils, we hypothesized that Pg exploits C5aR to subvert neutrophil defenses. The hypothesis was tested in vitro using human neutrophils and in vivo using the murine chamber model, in which >97% of the recruited leukocytes are neutrophils. Confocal microscopy and fluorescence resonance energy transfer showed that Pg comes into molecular proximity with a C5aR–Toll-like receptor (TLR)-2 complex in the lipid rafts of human neutrophils. Inhibition of either C5aR or TLR2 in vitro (human neutrophils) or in vivo (mice) promoted immune clearance of Pg, which was otherwise resistant to neutrophil killing. The intimate interaction of Pg with C5aR and its ability to activate this receptor without exogenously added C5a, suggested that Pg may bind directly C5aR. However, Pg was similarly susceptible to killing by C5aR- or C5deficient mice, suggesting a requirement for C5a in this evasive mechanism. Pg was unique among other tested oral bacteria to enzymatically generate C5a from C5 in the absence of complement
51 C5aR regulates intestinal microbiota composition and controls the induction of gastrointestinal allergic hypersensitivity Anna T. Reinicke 1 , Anne Herrmann 1 , Florian Baer 2 , Christian Sina 2 , Girish Srinivas 3,4 , Sven Kuenzel 3 , John Baines 3,5 , Jörg Köhl 1,6 1 Institute for Systemic Inflammation Research, University of Luebeck, 23538 Luebeck, Germany 2 Institute of Anatomy, University Hospital Schleswig-Holstein, Lübeck, Germany 3 Max Planck Institute for Evolutionary Biology, Ploen, Germany 4 Department of Dermatology, University of Luebeck, Lübeck, Germany 5 Institute for Experimental Medicine, University of Kiel, Kiel, Germany 6 Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital and University of Cincinnati, College of Medicine, Cincinnati, OH, USA
Allergic immunity provides important protection in host defense against non-infectious environmental toxins and irritants. However, when excessive, this defense may result in allergic disease. Complement C5a is known to control allergic responses in the lung but little is known about its role in allergic responses in the intestine. Here, we adopted a murine model of gastrointestinal allergy in which repeated oral challenge of ovalbumin (OVA) in OVA/alum-sensitized mice induced acute diarrhea. We observed that C5a receptor (C5aR) is required to mount an acute allergic response against oral allergen. Mechanistically, mast cell degranulation is a critical effector in mediating allergic diarrhea. We found that C5aR is crucial for the recruitment and activation of intestinal mucosal mast cells during gastrointestinal allergy by chloroacetate esterase staining of intestine paraffin sections and measurement of the release of the beta-chymase, MCPT-1. Furthermore, C5aR was found to be required for the production of allergen-specific immunoglobulins during allergy that are essential for mast cell-mediated degranulation. Genotype driven changes to gastrointestinal microbiota have been linked to diseases including inflammatory bowel disease (IBD) and allergic diseases. In this context, we examined the composition of bacterial microbiota in jejunum, ilium, caecum and colon of wild type and C5aR deficient littermates using 454 pyrosequencing. Our results show that the intestinal microbiota is significantly altered by C5aR whereby C5aR deficiency resulted in lower diversity of bacterial communities. Further, we observed a decreased frequency of Lactobacillus spp., which has been associated with protective immunity. We conclude that C5aR is an important determinant shaping the composition of the intestinal microbiota and influencing the ability to mount an allergic response in the intestine. http://dx.doi.org/10.1016/j.imbio.2012.08.052 52 C5aR-dependent cell activation by physiological concentrations of C5a-desArg: Insights from a novel label-free cellular assay Edimara S. Reis 1 , Hui Chen 1 , Georgia Sfyroera 2 , Peter N. Monk 3 , Jörg Köhl 4 , Daniel Ricklin 1 , John D. Lambris 1 1
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 National Center for Scientific Research “Democritos”, Athens, Greece 3 University of Sheffield, Sheffield, UK 4 University of Lübeck, Lübeck, Germany The complement anaphylatoxins C3a, C5a, and C5adesArg play critical roles in the induction of inflammation and the modulation
1147
of innate and acquired immune responses after binding to their G protein-coupled receptors, C3aR and C5aR. The role of C5adesArg in inducing cell activation has been often neglected, since the affinity of C5adesArg for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5adesArg and also C3a, C3adesArg and C5a to induce activation of transfected and primary immune cells. As expected, C3a and C5a, but not C3adesArg were able to induce cell activation. More importantly, our data indicate that physiological concentrations of C5adesArg can induce significant levels of cell activation that are even higher than that achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, since it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of Galpha-i , Galpha-s and Gbeta/gamma proteins upon C5aR engagement by C5a or C5adesArg . Further, mass spectrometric characterization of plasma-derived C5a and C5adesArg provided important insight into the post-translational modification pattern of these anaphylatoxins, which includes glycosylation at Asn64 and partial cysteinylation at Cys27. While the context-specific physiological contribution of C5adesArg has to be further explored, our data suggest that C5adesArg acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status. http://dx.doi.org/10.1016/j.imbio.2012.08.053 53 C5aR-dependent subversion of neutrophils and dysbiotic inflammation George Hajishengallis 1 , Jennifer L. Krauss 2 , Ravi Jotwani 2 , Toshiharu Abe 1 , Martha Triantafilou 3 , Kathy Triantafilou 3 , Robert A. DeAngelis 4 , John D. Lambris 4 1
University of Pennsylvania School of Dental Medicine, Department of Microbiology, Philadelphia, PA, USA 2 University of Louisville, Oral Health and Systemic Disease, Louisville, KY, USA 3 University of Cardiff, Institute of Infection & Immunity, Cardiff, UK 4 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Porphyromonas gingivalis (Pg) was recently shown to remodel the oral microbiota to a dysbiotic state that causes inflammatory periodontitis. Pg-induced dysbiosis requires a functional C5a receptor (C5aR), and the uncontrolled growth of the microbiota suggests a breakdown of immune surveillance in the periodontal tissue. Because >95% of the leukocytes recruited to the tooth-associated biofilm are neutrophils, we hypothesized that Pg exploits C5aR to subvert neutrophil defenses. The hypothesis was tested in vitro using human neutrophils and in vivo using the murine chamber model, in which >97% of the recruited leukocytes are neutrophils. Confocal microscopy and fluorescence resonance energy transfer showed that Pg comes into molecular proximity with a C5aR–Toll-like receptor (TLR)-2 complex in the lipid rafts of human neutrophils. Inhibition of either C5aR or TLR2 in vitro (human neutrophils) or in vivo (mice) promoted immune clearance of Pg, which was otherwise resistant to neutrophil killing. The intimate interaction of Pg with C5aR and its ability to activate this receptor without exogenously added C5a, suggested that Pg may bind directly C5aR. However, Pg was similarly susceptible to killing by C5aR- or C5deficient mice, suggesting a requirement for C5a in this evasive mechanism. Pg was unique among other tested oral bacteria to enzymatically generate C5a from C5 in the absence of complement