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Poster 20.9 Phosphorylcholine-modied macromolecules inhibit ATP-dependent inammasome activation in human monocytes Andreas Hecker, Mira Küllmar, Sigrid Wilker, Srebrena Atanasova, Marion Meixner (Laboratory of Experimental Surgery , Department of General and Thoracic Surgery, Justus-Liebig-University, Giessen, Germany), Thomas Timm (Institute of Biochemistry, Justus-Liebig- University, Giessen, Germany), Anna Zakrzewicz (Laboratory of Experimental Surgery , Department of General and Thoracic Surgery, Justus-Liebig-University, Giessen, Germany), Jochen Klein (Institute of Pharmacology, Johann-Wolfgang-Goethe University, Frankfurt, Germany), Winfried Padberg (Laboratory of Experimental Surgery , Department of General and Thoracic Surgery, Justus-Liebig-University, Giessen, Germany), Wolfgang Kummer (Institute of Anatomy and Cell Biology , Justus-Liebig-University, Giessen, Germany), Sabina Janciauskiene (Department of Respiratory Medicine , Hannover Medical School, Germany), Elke Schweda (Department of Physics, Chemistry and Biology, Linköping-University, Sweden), Günter Lochnit (Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany), Veronika Grau (Laboratory of Experimental Surgery , Department of General and Thoracic Surgery, Justus-Liebig-University, Giessen, Germany) *Members of the German Centre for Lung Research. Introduction: The release of bioactive interleukin-1β (IL-1β), a potent fever-inducing pro-inammatory cytokine, requires two independent danger signals. Prototypically, lipopolysaccharide (LPS, rst signal) induces synthesis of pro-IL-1β. Extracellular ATP (a classical second signal) is sensed by ATP receptor P2X7, induces inammasome activation, generation of active caspase-1, cleavage of pro-IL-1β, and rapid secretion of IL-1β. In parallel work, we demonstrated effective suppression of ATP-induced IL-1β secretion by ligands of nicotinic receptors containing subunit α9 (nAChRα9). Phosphorylcholine (PC)- modied macromolecules are produced by numerous pathogens and down-modulate diverse immune functions of the host. We postulate that PC-modied macromolecules are sensed by nAChRα9 and modulate ATP-induced inammasome activation. Methods: Cells of the human monocytic U937 cell line were primed with LPS from Escherichia coli and stimulated with BzATP (P2X7 agonist) or nigericine (bacterial pore-forming toxin) in the presence and absence of free PC, PC-modied bovine serum albumin (BSA), well characterized PC-modied LPS puried from two Haemophilus inuenzae wild type strains as well as with unmodied LPS from corresponding mutant strains defective in the biosynthesis of PC-LPS. IL-1β release was measured by ELISA. Results: Free PC, PC-modied BSA and PC-modied LPS dose- dependently inhibited BzATP-induced IL-1β release. Nigericine- induced secretion of IL-1β, however, was not impaired. Inhibition was antagonized by mecamylamine, a general nAChR antagonist, α-bungarotoxin, a specic antagonist of nAChR containing subunits α7 and α9, and strychnine, which antagonizes nAChRα9. In contrast to their PC-modied counterparts, unmodied BSA and LPS from mutant bacteria were not effective. Discussion: We propose a novel, highly effective mechanism of immune evasion involving suppression of ATP-induced IL-1β release via nAChR containing subunit α9 by a broad range of PC-modied pathogens and pathogen-derived secretory products. PC-BSA is a valid model compound mimicking PC-modied immunomodulatory pro- teins secreted by eukaryotic parasites, and PC-modied bacterial wall components facilitate the colonization of the host. Funding: LOEWE NNCS. doi:10.1016/j.autneu.2013.08.060 Poster 20.12 ATP-mediated release of interleukin-1β is inhibited by activation of nicotinic acetylcholine receptors containing subunit α9 Mira Küllmar, Andreas Hecker (Laboratory of Experimental Surgery*, Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Germany), Katrin Richter (Institute of Animal Physiology, Justus-Liebig-University, Giessen, Germany), Sigrid Wilker, Srebrena Atanasova, Anna Zakrzewicz, Sabine Stumpf (Laboratory of Experimental Surgery*, Department of General and Thoracic Surgery, Justus-Liebig- University Giessen, Germany), Andreas Kaufmann, Stefan Bauer (Insti- tute of Immunology*, Philipps-University, Marburg, Germany), Wolfgang Kummer (Institute for Anatomy and Cell Biology*, Justus-Liebig- University, Giessen, Germany), Winfried Padberg (Laboratory of Exper- imental Surgery*, Department of General and Thoracic Surgery, Justus- Liebig-University Giessen, Germany), Martin Fronius (Institute of Animal Physiology, Justus-Liebig-University, Giessen, Germany), Veronika Grau (Laboratory of Experimental Surgery*, Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Germany) *Members of the German Centre for Lung Research. Introduction: Secretion of the pro-inammatory monokine interleukin- 1β (IL-1β) is tightly controlled to prevent excessive fever and tissue damage. Priming of mononuclear phagocytes with lipopolysaccharide (LPS) induces pro-IL-1β synthesis. A second signal such as extracellular ATP leads to inammasome activation, activation of caspase-1, cleavage of pro-IL-1β, and a rapid release of bioactive IL-1β. Previously, we demonstrated that acetylcholine produced by mononuclear leukocytes activated by acute allograft rejection down-modulates calcium signals in response to ATP (Hecker et al., J Leukoc Biol 2007). Here, we test the hypothesis that acetylcholine inhibits ATP-mediated inammasome activation. Methods: LPS-primed monocytic U937 cells, freshly isolated naïve human peripheral blood mononuclear leukocytes (PBMC) and PBMC isolated from the vasculature of rat renal allografts during acute rejection were stimulated with BzATP (P2X7 agonist) or nigericine (bacterial pore-forming toxin) in the presence and absence of cholinergic agonists and antagonists. IL-1β release was measured by ELISA and patch clamp technology was used to monitor ion currents in U937 cells. Results: Acetylcholine, choline and nicotine dose-dependently inhibited BzATP- but not nigericine-induced release of IL-1β from LPS-primed U937 cells and naïve PBMC. Inhibition was antagonized by mecamyl- amine, a general antagonist of nicotinic acetylcholine receptors (nAChR), α-bungarotoxin, a specic antagonist of nAChR containing subunits α7 and α9, and strychnine, which antagonizes nAChRα9. BzATP-induced ion currents were abolished by nicotine, but restored by mecamylamine. Activated allograft PBMC did not release IL-1β in response to BzATP but did so in the presence of exogenous acetylcholine esterase. Conclusions: We suggest a novel anti-inammatory cholinergic mechanism in monocytes, which is mediated by nAChR containing subunit α9. AChR activation potently inhibits signalling via P2X7 receptors upstream of inammasome activation and eventually IL-1β release. This mechanism seems to be effective in vivo in activated allograft PBMC, where endogenous acetylcholine suppresses secre- tion of IL-1β in response to ATP. Abstracts / Autonomic Neuroscience: Basic and Clinical 177 (2013) 297319 318

ATP-mediated release of interleukin-1β is inhibited by activation of nicotinic acetylcholine receptors containing subunit α9

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Page 1: ATP-mediated release of interleukin-1β is inhibited by activation of nicotinic acetylcholine receptors containing subunit α9

Poster 20.9

Phosphorylcholine-modifiedmacromolecules inhibit ATP-dependentinflammasome activation in human monocytes

Andreas Hecker, Mira Küllmar, Sigrid Wilker, Srebrena Atanasova,Marion Meixner (Laboratory of Experimental Surgery

, Departmentof General and Thoracic Surgery, Justus-Liebig-University, Giessen,Germany), Thomas Timm (Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany), Anna Zakrzewicz (Laboratory ofExperimental Surgery

, Department of General and Thoracic Surgery,Justus-Liebig-University, Giessen, Germany), Jochen Klein (Instituteof Pharmacology, Johann-Wolfgang-Goethe University, Frankfurt,Germany), Winfried Padberg (Laboratory of Experimental Surgery

,Department of General and Thoracic Surgery, Justus-Liebig-University,Giessen, Germany), Wolfgang Kummer (Institute of Anatomy andCell Biology

, Justus-Liebig-University, Giessen, Germany), SabinaJanciauskiene (Department of Respiratory Medicine

, Hannover MedicalSchool, Germany), Elke Schweda (Department of Physics, Chemistry andBiology, Linköping-University, Sweden), Günter Lochnit (Institute ofBiochemistry, Justus-Liebig-University, Giessen, Germany), VeronikaGrau (Laboratory of Experimental Surgery

, Department of General andThoracic Surgery, Justus-Liebig-University, Giessen, Germany)

*Members of the German Centre for Lung Research.

Introduction: The release of bioactive interleukin-1β (IL-1β), a potentfever-inducing pro-inflammatory cytokine, requires two independentdanger signals. Prototypically, lipopolysaccharide (LPS, first signal)induces synthesis of pro-IL-1β. Extracellular ATP (a classical secondsignal) is sensed by ATP receptor P2X7, induces inflammasomeactivation, generation of active caspase-1, cleavage of pro-IL-1β, andrapid secretion of IL-1β. In parallel work, we demonstrated effectivesuppression of ATP-induced IL-1β secretion by ligands of nicotinicreceptors containing subunit α9 (nAChRα9). Phosphorylcholine (PC)-modified macromolecules are produced by numerous pathogens anddown-modulate diverse immune functions of the host. We postulatethat PC-modified macromolecules are sensed by nAChRα9 andmodulate ATP-induced inflammasome activation.

Methods: Cells of the human monocytic U937 cell line were primedwith LPS from Escherichia coli and stimulated with BzATP (P2X7agonist) or nigericine (bacterial pore-forming toxin) in the presenceand absence of free PC, PC-modified bovine serum albumin (BSA),well characterized PC-modified LPS purified from two Haemophilusinfluenzae wild type strains as well as with unmodified LPS fromcorresponding mutant strains defective in the biosynthesis of PC-LPS.IL-1β release was measured by ELISA.

Results: Free PC, PC-modified BSA and PC-modified LPS dose-dependently inhibited BzATP-induced IL-1β release. Nigericine-induced secretion of IL-1β, however, was not impaired. Inhibitionwas antagonized by mecamylamine, a general nAChR antagonist,α-bungarotoxin, a specific antagonist of nAChR containing subunitsα7 and α9, and strychnine, which antagonizes nAChRα9. In contrastto their PC-modified counterparts, unmodified BSA and LPS frommutant bacteria were not effective.

Discussion: We propose a novel, highly effective mechanism ofimmune evasion involving suppression of ATP-induced IL-1β releasevia nAChR containing subunit α9 by a broad range of PC-modifiedpathogens and pathogen-derived secretory products. PC-BSA is a validmodel compound mimicking PC-modified immunomodulatory pro-teins secreted by eukaryotic parasites, and PC-modified bacterial wallcomponents facilitate the colonization of the host.

Funding: LOEWE NNCS.

doi:10.1016/j.autneu.2013.08.060

Poster 20.12

ATP-mediated release of interleukin-1β is inhibited by activationof nicotinic acetylcholine receptors containing subunit α9

Mira Küllmar, Andreas Hecker (Laboratory of Experimental Surgery*,Department of General and Thoracic Surgery, Justus-Liebig-UniversityGiessen, Germany), Katrin Richter (Institute of Animal Physiology,Justus-Liebig-University, Giessen, Germany), Sigrid Wilker, SrebrenaAtanasova, Anna Zakrzewicz, Sabine Stumpf (Laboratory of ExperimentalSurgery*, Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Germany), Andreas Kaufmann, Stefan Bauer (Insti-tute of Immunology*, Philipps-University,Marburg, Germany),WolfgangKummer (Institute for Anatomy and Cell Biology*, Justus-Liebig-University, Giessen, Germany), Winfried Padberg (Laboratory of Exper-imental Surgery*, Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Germany),Martin Fronius (Institute of AnimalPhysiology, Justus-Liebig-University, Giessen, Germany), Veronika Grau(Laboratory of Experimental Surgery*, Department of General andThoracic Surgery, Justus-Liebig-University Giessen, Germany)

*Members of the German Centre for Lung Research.

Introduction: Secretion of thepro-inflammatorymonokine interleukin-1β (IL-1β) is tightly controlled to prevent excessive fever and tissuedamage. Priming of mononuclear phagocytes with lipopolysaccharide(LPS) induces pro-IL-1β synthesis. A second signal such as extracellularATP leads to inflammasome activation, activation of caspase-1, cleavageof pro-IL-1β, and a rapid release of bioactive IL-1β. Previously, wedemonstrated that acetylcholine produced by mononuclear leukocytesactivated by acute allograft rejection down-modulates calcium signals inresponse to ATP (Hecker et al., J Leukoc Biol 2007). Here, we test thehypothesis that acetylcholine inhibits ATP-mediated inflammasomeactivation.

Methods: LPS-primed monocytic U937 cells, freshly isolated naïvehuman peripheral blood mononuclear leukocytes (PBMC) and PBMCisolated from the vasculature of rat renal allografts during acute rejectionwere stimulated with BzATP (P2X7 agonist) or nigericine (bacterialpore-forming toxin) in the presence and absence of cholinergic agonistsand antagonists. IL-1β release was measured by ELISA and patch clamptechnology was used to monitor ion currents in U937 cells.

Results:Acetylcholine, choline and nicotine dose-dependently inhibitedBzATP- but not nigericine-induced release of IL-1β from LPS-primedU937 cells and naïve PBMC. Inhibition was antagonized by mecamyl-amine, a general antagonist of nicotinic acetylcholine receptors (nAChR),α-bungarotoxin, a specific antagonist of nAChR containing subunits α7and α9, and strychnine, which antagonizes nAChRα9. BzATP-inducedion currentswere abolished by nicotine, but restored bymecamylamine.Activated allograft PBMC did not release IL-1β in response to BzATP butdid so in the presence of exogenous acetylcholine esterase.

Conclusions: We suggest a novel anti-inflammatory cholinergicmechanism in monocytes, which is mediated by nAChR containingsubunit α9. AChR activation potently inhibits signalling via P2X7receptors upstream of inflammasome activation and eventually IL-1βrelease. This mechanism seems to be effective in vivo in activatedallograft PBMC, where endogenous acetylcholine suppresses secre-tion of IL-1β in response to ATP.

Abstracts / Autonomic Neuroscience: Basic and Clinical 177 (2013) 297–319318

Page 2: ATP-mediated release of interleukin-1β is inhibited by activation of nicotinic acetylcholine receptors containing subunit α9

Funding: LOEWE NNCS.

doi:10.1016/j.autneu.2013.08.061

Poster 20.13

The influence of new titanium alloys and factors of thenon-neuronal cholinergic system (ACh, nicotine) as wellas the neutotrophine BDNF on human multipotent stromacells of osteoporotic bone in vitro

V. Kauschke, O. Dakischew (Laboratory for Experimental TraumaSurgery, JLU, Giessen, Germany), A. Helth, A. Gebert (Leibniz Institutefor Solid State and Materials Research, Dresden, Germany), C. Heiss,V. Alt, G. Schlewitz (Department of Trauma Surgery, University Hospitalof Giessen-Marburg, Germany), I. Panzer (Laboratory for ExperimentalTrauma Surgery, JLU, Giessen, Germany), O. Kilian (Laboratory forExperimental Trauma Surgery, JLU, Giessen, Germany; Central ClinicBad Berka, Germany), R. Schnettler (Laboratory for ExperimentalTrauma Surgery, JLU, Giessen, Germany; Department of TraumaSurgery, University Hospital of Giessen-Marburg, Germany), K.S. Lips(Laboratory for Experimental Trauma Surgery, JLU, Giessen, Germany)

Osteoporosis is declared by WHO as one of the 10 most importantand cost intensive diseases worldwide. It is characterized by adiminished bone mineral density and an increase in fracture risk. Thecommercial available bone substitution materials are not suitable forosteoporotic bone. Often bones break next to implant materials.

Recently, it has been shown that administration of nicotine to humanosteoblastic cells growing on plasma-sprayed commercial titaniumimplants increased cell proliferation, alkaline phosphate (ALP) activityand stimulated the matrix mineralization (Pereira et al. 2009, J BiomedMater Res A. 88, 84–93). In contrary to this findings others reportednicotine in vivo was detrimental to bone (Hapidin et al. 2007, J BoneMiner Metab 25, 93–98).

Therefore we asked whether acetylcholine, nicotine and thebrain-derived neurotrophic factor (BDNF) are involved in differen-tiation and proliferation of osteoblasts at the interface of titanium40 Nb which was additionally modified by grinding and etching. Weaddressed this issue by means of live cell observation, proliferation(LDH) and differentiation (ALP) assays.

Our preliminary results indicate that titanium 40 Nb showed goodbiocompatibility on hMSC in vitro. Proliferation of hMSC was strongerup-regulated in the osteoporotic donor than in the healthy one,regardless of the titanium modification tested. Nicotine showed lesseffect on the proliferation of hMSC but increased time dependently theALP activity and therefore the osteoblastic differentiation which wasgenerally higher in the non-osteoporotic donor. ACh and BDNF seemedto stimulate and accelerate differentiation as well as proliferation ofhMSC. This effect was lower for ground titanium. However these resultshave to be confirmed by further experiments.

Funded by DFG SFB/TRR 79.

doi:10.1016/j.autneu.2013.08.062

Poster 20.16

Evaluation of suitability of acetylcholine receptor antibodies forimmune detection in peripheral tissues

Frank Risto Rommel, Badrinarayanan Raghavan (PsychoneuroimmunologyLaboratory, Department of Psychosomatic Medicine and Psychotherapy,Justus-Liebig-University, Giessen, Germany), Renate Paddenberg (Instituteof Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany),Susanne Tumala (Psychoneuroimmunology Laboratory, Department ofPsychosomatic Medicine and Psychotherapy, Justus-Liebig-University,Giessen, Germany), Uwe Gieler (Department of Dermatology, UniversityHospital Giessen, Giessen, Germany), Eva M.J. Peters (Psychoneuroim-munology Laboratory, Department of Psychosomatic Medicine andPsychotherapy, Justus-Liebig-University, Giessen, Germany)

Tremendous progress in research over the past few years haswidened the horizons in the understanding of non-neuronal cholinergicsignaling with increasing evidences on acetylcholine and its receptorshaving a crucial role in the regulation of peripheral inflammatorydiseases. Specifically, muscarinic receptor 3 (MR3) and nicotinicreceptor α7 (α7nAChR) may have a profound role in the stressadaptation in allergic skin inflammation. However, reliable localizationstudies are necessary to fully elucidate the distinct roles of thesereceptors with respect to stress and inflammatory response. From theliterature, it is evident that most of the available MR3 and α7nAChRantibodies have a high degree of unspecificity. Therefore we evaluatedthe specificity of these acetylcholine receptor antibodies using immu-nohistochemical labeling of mouse back skin sections from wild-type(wt) and corresponding gene-deficient (KO) animals. 3 new commer-cially available antibodies for MR3 and 4 new antibodies for α7nAChRwere tested, focusing on positive staining of mast cells, nerve fibers andkeratinocytes as outcome parameters. In the case of M3R, all the 3antibodieswere not giving any difference in staining ofmast cells, nervefiber and keratinocytes between the wt and KO animals, suggesting anunspecific staining pattern. The first α7nAChR antibody employed wasraised against the first 100 amino acids (undeleted region in KO mice)of the α7nAChR and gave positive staining in all investigated cellpopulations in skin of wt micewhereas unexpectedly nerve fibers wereexclusively unlabeled in the KO mice suggesting unspecific staining inmast cells and keratinocytes. The next 3 antibodies used were raisedagainst c-terminal end of α7nAChR (exons 8–10 are deleted in the KOmice), of which one antibody showed no staining in the KO animalwhile the other two antibodies produced unspecific staining. Interest-ingly, the first antibody produced a dramatically reduced western blotsignal in skin but not brain of KO mice and RT-PCR analysis revealedmRNA amplification with a primer set for the undeleted region in bothWT and α7nAChR KO mouse skin but none with a primer set for thedeleted region in KOmice. On the basis of our newdata,we suggest thatthere may be an expression of an additional, potentially truncatedvariant of α7nAChR in non-neuronal peripheral tissues. However, thefunctionality of these variants must be addressed especially in theregulation of immune function of the skin.

doi:10.1016/j.autneu.2013.08.063

Abstracts / Autonomic Neuroscience: Basic and Clinical 177 (2013) 297–319 319