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ABSTRACT

BOOK

32nd Annual Conference

The cross-road between macrophages and dendritic cells: from immunometabolism to single cell fate

EMDS2018

Polo Didattico “Giorgio Zanotto” - Verona, Italy27-29 September, 2018

32nd Annual Conference Polo Didattico “Giorgio Zanotto”Verona, Italy

27-29 September, 2018


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INDEXCommitees .............................................................................................................................................. 2

Welcome .................................................................................................................................................. 3

Abstracts Index ....................................................................................................................................... 4

Oral Communication Abstracts ............................................................................................ 11

Poster Abstracts ...................................................................................................................... 24

Authors’ Index ...................................................................................................................................... 85

COMMITEESScientific Organizers Prof. Vincenzo Bronte (University of Verona)Prof. Ursula Grohmann (University of Perugia)

Scientific Secretariat Dr. Susanna Mandruzzato (University of Padova)Dr. Giada Mondanelli (University of Perugia)Dr. Silvia Sartoris (University of Verona)Dr. Stefano Ugel (University of Verona)

Local Secretariat Mr. Daniel Lovato (Hospital Trust of Verona)

The Council of EMDS Prof. Thomas Decker (University of Vienna)Prof. Jonathan Jantsch (University of Regensburg)Prof. Amaya Puig Kröger (Hospital General Universitario Gregorio Marañón, Madrid)Geert Raes (University of Brussels)Maciek Siedlar (Jagiellonian University, Krakow)Ulrike Schleicher (University of Erlangen)




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Dear Colleagues,we are very proud to welcome you to Verona (Italy) for the 32nd Annual Conference of European Macrophage & Dendritic Cell Society (EMDS) entitled The cross-talk between macrophages and dendritic cells: from immunometabolism to single cell fate. The conference will take place on 27-29 September, 2018 at Polo Didattico “G. Zanotto” – Verona, Italy

Myeloid cells comprise different populations of mature, terminally differentiated macrophages, dendritic cells and neutrophils, as well as immature myeloid progenitors. These cells are evolutionarily designed to protect the host from pathogens, such as bacteria and viruses. However, during chronic inflammation-associated diseases, such as cancer, these cells undergo sequential phenotypical changes linked to an alteration of the normal hematopoiesis that promotes the expansion of myeloid cells with negative immune regulatory activity on adaptive immunity.

The specific goals of this meeting are a better understanding of myeloid cells ontogeny, under normal and pathological conditions, a critical analysis of markers and genetic signatures that can partially resolve the definition of their identity, and potential therapeutic approaches able to increase the efficacy of current immunotherapy.

The program is based on 6 specific sections in which 19 excellent scientists will share their exciting research results with us. Moreover, we invited 4 outstanding scientists, whose studies had deeply imprinted the current knowledge about myeloid cells, to deliver a specific keynote lecture during the congress. Finally, we will have a lively poster session providing a great occasion to discuss and socialize with colleagues and we will select 12 abstracts presented by young researchers as short talks. Besides an outstanding scientific program, our meeting will provide opportunities for participants to meet colleagues, build networks, and establish future collaborations.

Therefore, we are very excited to offer this incredible opportunity and are looking forward to an active and professional networking, in addition to vigorous discussion of cutting edge science in the myeloid cell field.

The 2018 Scientific Program Chairs

Vincenzo Bronte, M.D. University of Verona

Ursula Grohmann, Ph.D. University of Perugia




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ABSTRACTS INDEXOC1/1 SMALL MOLECULE MODULATORS OF PRION PROTEIN (PRPC) IN DENDRITIC CELLS REGULATE

INFLAMMATORY RESPONSES IN A MODEL OF MULTIPLE SCLEROSIS Giorgia Manni - M. Letizia Barreca - Giulia Scalisi - Emiliano Basini - Marco Gargaro - Giuseppe

Manfroni - Francesca Fallarino ................................................................................................................ 11

OC2/1 NEUTROPHIL TRAPS IN LEUKEMIA: FROM TRIGGERS OF DISEASE PROGRESSION TO VEHICLE FOR NEW VACCINES

Sabina Sangaletti - Claudio Tripodo - Mario Paolo Colombo ............................................................ 12

OC3/2 IL23 SECRETED BY MYELOID CELLS DRIVES CASTRATION RESISTANT PROSTATE CANCER Arianna Calcinotto - Clarissa Spataro - Elena Zagato - Johann De Bono - Andrea Alimonti ......... 13

OC4/2 CCR1 AND CCR5 CONTROL THE POLARIZATION COMMITMENT OF EARLY MYELOID PRECURSORS IN TUMOR HOST

Serena Zilio - Donald T. Weed - Alessia Zoso - Dimitri Van Simaeys - Carla Rodriguez - Emilia Mazza

- Silvio Bicciato - Vincenzo Bronte - Paolo Serafini .............................................................................. 14

OC5/3 GLUCOCORTICOIDS IMPAIR PHAGOCYTOSIS AND INFLAMMATORY RESPONSE IN A MODEL OF HUMAN MACROPHAGES

Mauricio Olivares-Morales - Marjorie De la Fuente - Karen Dubois - Daniela Parada - David Diaz-Jiménez - Xiaojiang Xu - Nayaret Chamorro - Rodrigo Quera - John Cidlowski - Roberto Vidal - Marcela A. Hermoso ................................................................................................................................ 15

OC6/3 CD16+ MONOCYTES ARE PRECURSORS FOR DENDRITIC CELLS WITH RALDH ACTIVITY THAT PROMOTE PATHOGENICITY DURING HIV-1 INFECTION. 1

Amélie Cattin - Vanessa Sue Wacleche - Natalia Fonseca Do Rosario - Jean-Philippe Goulet - Dominique Gauchat - Annie Gosselin - Jean-Pierre Routy - Petronela Ancuta ............................... 16

OC7/4 MAPPING THE HUMAN IMMUNE LANDSCAPE IN THE ALVEOLAR SPACE OF HEALTHY DONORS AND COPD PATIENTS

Kevin Baßler - Wataru Fujii - Theodoros Kapellos - Carmen Pizarro - Drik Skowasch - Joachim Schultze ..................................................................................................................................................... 17

OC8/4 INTEGRATION OF IMMUNE STIMULI AT THE SINGLE-CELL LEVEL EXPANDS THE SPECTRUM OF MACROPHAGE ACTIVATION STATES AND IDENTIFIES TARGETS FOR FUNCTIONAL REPROGRAMMING

Marco Genua - Francesco Cilenti - Giulia Barbiera - Dario Iodice - Elisa Montaldo - Eleonora Lusito - Renato Ostuni .......................................................................................................................................... 18

OC9/4 SINGLE-CELL CHARACTERIZATION OF ADIPOSE TISSUE MACROPHAGES André Sulen - Emelie Barreby - Francisco Verdeguer - Camille Bleriot - Florent Ginhoux - Myriam

Aouadi ...................................................................................................................................................... 19

OC10/5 MTOR SIGNALING IN MACROPHAGES CONTRIBUTES TO THE MAINTENANCE OF IRON HOMEOSTASIS Nyamdelger Sukhbaatar - Monika Linke - Maria Schöller - Igor Theurl - Günter Weiss - Stephanie

Deborah Fritsch - Stefanie Horer - Barbara Scheiber-mojdehkar - Markus Hengstschläger - Thomas Weichhart ................................................................................................................................................ 20

OC11/5 DYNAMIC AGE-DEPENDENT CHANGES IN THE DEVELOPMENTAL ORIGIN OF MONONUCLEAR PHAGOCYTES IN THE KIDNEY

Natallia Salei - Stephan Rambichler - Nikolaos Papaioannou - Johanna Salvermoser - Ronja Schuchert

- Filippo Cernilogar - Gunnar Schotta - Christian Schulz - Barbara U. Schraml .............................. 21




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OC12/6 IL10 POLARIZATION REGULATES TNF-SIGNALING AND MITOCHONDRIAL FUNCTION IN HUMAN MACROPHAGES

Hung-jen Chen - Guillermo Griffith - Menno De Winther .................................................................. 22

OC13/6 SEMAPHORIN 6D REVERSE SIGNALING CONTROLS MACROPHAGE LIPID METABOLISM AND ANTI-INFLAMMATORY POLARIZATION

Sujin Kang - Atsushi Kumanogoh .......................................................................................................... 23

P1 ISOLATION AND CHARACTERIZATION OF FUNCTIONAL CROSS-PRESENTING DENDRITIC CELLS FROM MOUSE TISSUES AND TUMOR

Frank Ralph Morrissey-Wettey - Cristina Conforti Andreoni - Daniela Vorholt - Andrzej Dzionek ... 24

P2 THE ROLE OF COAGULATION FACTORS EXPRESSED ON MONOCYTE DERIVED MACROPHAGES Hannes Datler - Omar Sharif - Andrea Vogel - Martina Keindl - Julia Brunner - Melanie Hofmann -

Laszlo Musiejovsky - Christina Baumgartinger - Jose Basilio - Georg Obermayer - Christoph J. Binder

- Gernot Schabbauer ............................................................................................................................... 25

P3 EVALUATION OF GM3-CONTAINING LIPOSOMES FOR ANTIGEN TARGETING TO SPLENIC CD169+ MACROPHAGES TO INDUCE ANTI-CANCER IMMUNITY

Joanna Grabowska - Dieke Van Dinther - Katarzyna Olesek - Leoni Hoogterp - Martino Ambrosini - Paul R. Crocker - Gert Storm - Yvette Van Kooyk - Joke M.m. Den Haan ........................................ 26

P4 AMPLIFIED HOST DEFENSE BY TOLL-LIKE RECEPTOR-MEDIATED DOWNREGULATION OF THE GLUCOCORTICOID-INDUCED LEUCINE ZIPPER (GILZ) IN MACROPHAGES

Jessica Hoppstädter - Britta Diesel - Nina Hachenthal - Marie Minet - Petra Leidinger - Christina Backes

- Friedrich Grässer - Eckart Meese - Stefano Bruscoli - Carlo Riccardi - Hanno Huwer - Alexandra K. Kiemer ...................................................................................................................................................... 27

P5 THE ROLE OF DENDRITIC CELL SUBSETS IN T REGULATORY CELL INDUCTION DURING ALLERGIC AIRWAY INFLAMMATION

Emma Houlder - Sheila Brown - Amanda Ridley - Freya Svedberg - Cecilia Forss - Mark Wilson - Antoon Van Oosterhout - Matthew Edwards - Peter Cook - Andrew Macdonald ............................. 28

P6 PTEN IN APCS REGULATES AUTOIMMUNITY BY T-CELL CHECK POINT INHIBITION Martina Kerndl - Andrea Vogel - Melanie Hofmann Julia Stefanie Brunner - Hannes Datler - Omar

Sharif - Gernot Schabbauer .................................................................................................................... 29

P7 THE E3 UBIQUITIN LIGASE PELLINO2 MEDIATES TLR9-INDUCED INFLAMMATION Ewa Oleszycka - Ronan Bergin - Paul N. Moynagh ............................................................................. 30

P8 HUMAN PERIPHERAL BLOOD MONOCYTES EXCEED MONOCYTE-DERIVED MACROPHAGES AS PROSTAGLANDIN SOURCES

Sonja Rittchen - Katharina Jandl - Rufina Schuligoi Akos Heinemann ............................................. 31

P9 RAPAMYCIN INHIBITS THE EXPRESSION OF PROINSULIN CELLS IN BONE MARROW DERIVED DENDRITIC CELLS FROM DIABETIC PATIENTS. AN IN VITRO STUDY

Luisa Sambado - Piero Maria Stefani - Laura Valori - Michele Gottardi - Lucia Zanatta - Matilde Cacciatore - Angelo Paolo Dei Tos - Agostino Paccagnella - Maria Sambataro ................................. 32

P10 ROLE OF COMPLEMENT INHIBITOR FACTOR H IN MONOCYTE PHAGOCYTOSIS Karolina Izabela Smolag - Anna Maria Blom - Myriam Martin ....................................................... 33




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P11 CELLULAR AND VIRAL FLIP PROGRAMS BROAD IMMUNE REGULATORY TRANSCRIPTION IN MONOCYTES

Alessandra Fiore - Stefano Ugel - Francesco De Sanctis - Sara Sandri - Giulio Fracasso - Rosalinda Trovato - Silvia Sartoris - Samantha Solito - Susanna Mandruzzato - Fulvia Vascotto - Keli Hippen - Giada Mondanelli - Ursula Grohmann - Geny Piro - Carmine Carbone - Davide Melisi - Rita Lawlor - Aldo Scarpa - Alessia Lamolinara - Manuela Iezzi - Matteo Fassan Silvio Bicciato - Bruce Blazar - Ugur Sahin - Peter Murray - Vincenzo Bronte .................................................................................. 34

P12 DISCOVERING NOVEL IMMUNE-MODULATORY GLYCANS USING HIGH-THROUGHPUT SCREENING STRATEGIES

Meshal Alobaid - Sarah-jane Richards - Mathew Gibbson - Morgan Alexander - Amir Ghaemmaghami .................................................................................................................................. 35

P13 MACROPHAGE TARGETING BY THE CYTOTOXIC NATURAL PRODUCT THIOHOLGAMIDE A AS AN ANTICANCER PRINCIPLE

Charlotte Dahlem - Maria Lopatniuk - Jessica Hoppstädter - Sonja M. Kessler - Britta Diesel - Andriy Luzhetskyy - Alexandra K. Kiemer ..................................................................................................... 36

P14 HYPERTHERMIC TREATMENT AT 56°C INDUCES TUMOR-SPECIFIC IMMUNE PROTECTION IN A MOUSE MODEL OF PROSTATE CANCER IN BOTH PROPHYLACTIC AND THERAPEUTIC IMMUNIZATION REGIMENS

Francesco De Sanctis - Sara Sandri - Matteo Martini - Marta Mazzocco - Alessandra Fiore - Rosalinda Trovato - Stefano Garetto - Davide Brusa - Stefano Ugel - Silvia Sartoris ....................................... 37

P15 REPROGRAMMING OF TUMOR-ASSOCIATED MACROPHAGES THROUGH ID4-DEPENDENT PARACRINE ACTIVITY OF BREAST CANCER CELLS

Sara Donzelli - Elisa Milano - Magdalena Pruszko - Andrea Sacconi - Silvia Masciarelli - Enzo Gallo - Alicja Zylicz - Francesco Fazi - Giovanni Blandino - Giulia Fontemaggi ......................................... 38

P16 ADENOSINE REPOLARISES HUMAN MACROPHAGES TO AN IMMUNOSUPPRESSIVE PHENOTYPE VIA BOTH A2A AND A2B ADENOSINE RECEPTORS

A.M. Jackson - A.P. Kaur - T. Musarrat - P.M. Patel - S.J. Hill - H.A. Franks ................................... 39

P17 MYELOID MTORC1 AND ITS INVOLVEMENT IN COLITIS AND COLORECTAL CANCER Stephanie Deborah Fritsch - Birgit Schütz - Nyamdelger Sukbhbaatar - Michaela Lang - Christoph

Magnes - Jakob Weiszmann - Markus Hengstschläger - Thomaas Weichhart .................................. 40

P18 DEFINING THE IMMUNOMODULATORY EFFECTS OF IMM-101: A PROMISING, NOVEL CO-THERAPY FOR CANCER

Alicia Galdon - James Crooks - Sheila Brown - Laura Rosa Brunet - Andrew Macdonald .............. 41

P19 INACTIVATION OF MTORC2 IN MACROPHAGES IS A SIGNATURE OF COLORECTAL CANCER THAT PROMOTES TUMORIGENESIS

Karl Katholnig - Birgit Schütz - Stephanie Deborah Fritsch - David Schörghofer - Monika Linke - Nyamdelger Sukhbaatar - Julia Maria Matschinger - Daniela Unterleuthner - Martin Hirtl - Michaela Lang - Merima Herac - Andreas Spittler - Andreas Bergthaler - Gernot Schabbauer - Helmut Dolznig

- Mark A. Magnuson - Mario Mikula - Stefanie Horer - Thomas Weichhart ................................... 42

P20 CHARACTERIZATION OF A NEW IN VITRO MODEL FOR TUMOR-ASSOCIATED MACROPHAGES AND COMPARISON WITH PATIENT-DERIVED MACROPHAGES FROM LUNG TUMOR TISSUE

Jessica Hoppstädter - Anna Dembek - Nathalie Wirth - Daniela Oster - Gil Gasparoni - Martin Simon - Marcel H. Schulz - Hanno Huwer - Alexandra K. Kiemer ................................................................ 43




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P21 NEW MONOCYTE ATTRACTING AND PRO-ANGIOGENIC FACTOR YKL-39 IS PRODUCED BY TUMOR-ASSOCIATED MACROPHAGES IN HUMAN BREAST CANCER AND IS INDICATIVE FOR INCREASED METASTASIS

Tengfei Liu - Irina Larionova - Vladimir Riabov - Nikolai Litviakov - Marina Zavyalova - Andrew Flatley - Elisabeth Kremmer - Nadezhda Cherdyntseva - Harald Klüter - Julia Kzhyshkowska ...... 44

P22 THE CHEMOKINE CCL17 SHAPES THE TUMOR MICROENVIRONMENT IN MULTIPLE MOUSE MODELS OF CANCER

Rebecca Metzger - Anne Krug ............................................................................................................. 45

P23 TRANSCRIPTOMICS LANDSCAPE OF NECROPTOSIS GENES IS ASSOCIATED WITH DENDRITIC CELLS INFILTRATION: A PAN-CANCER STUDY OF 5,451 PRIMARY SOLID TUMORS

Lorenzo Nicolè - Tiziana Sanavia - Rocco Cappellesso - Ambrogio Fassina .................................... 46

P24 MACROPHAGES ADAPT TO THE METASTATIC NICHE IN PANCREATIC CANCER BY SENSING THE STIFFNESS

Sebastian Nielsen - Edward Horton - Giorgia Maltese - Chang-il Hwang - James Mcconnell - Michael Sherratt - Janine Erler ......................................................................................................................... 47

P25 LEUKOCYTE-MIMETIC NANOPARTICLES TO TARGET INFLAMMATION Anna Pasto - Manuela Sushnitha - Jonathan O. Martinez - Roberto Molinaro - Ennio Tasciotti ... 48

P26 CHARACTERIZATION OF CIRCULATING AND TUMOR-INFILTRATING MYELOID-DERIVED SUPPRESSOR CELLS IN PATIENTS AFFECTED BY PANCREATIC DUCTAL ADENOCARCINOMA

Sara Sartori - Rosalinda Trovato - Alessandra Fiore - Francesco De Sanctis - Rosalba Giugno - Luciano Cascione - Salvatore Paiella - Francesca Hofer - Ornella Poffe - Cristina Anselmi - Tiziana Cestari - Samantha Solito - Susanna Mandruzzato - Boris Rusev - Silvia Sartoris - Rita Teresa Lawlor

- Aldo Scarpa - Claudio Bassi - Vincenzo Bronte - Stefano Ugel ....................................................... 49

P27 REGULATION OF ARGINASE1 AND INDUCIBLE NITRIC OXIDE SYNTHASE IN SALMONELLA TYPHIMURIUM INFECTED BONE MARROW DERIVED MACROPHAGES

Natascha Brigo - Stefanie Dichtl - Christa Pfeifhofer-Obermair - Piotr Tymoszuk - Markus Seifert - Günter Weiss ....................................................................................................................................... 50

P29 AN IN VITRO APPROACH TO STUDY MACROPHAGE HYPERPLASIA DURING P. FALCIPARUM MALARIA Jill Dalimot - Thomas Klei - Ryanne Arisz - Judy Geissler - Guillaume Bouyer - Stéphane Egée - Robin

Van Bruggen ....................................................................................................................................... 51

P30 REPEATED MYCOBACTERIA VACCINATION IN MICE INDUCES MYELOID-DERIVED SUPPRESSOR CELL KILLING OF SPLENIC DENDRITIC CELLS VIA INOS-DEPENDENT NO PRODUCTION

Eliana Ribechini - Ina Eckert - Andreas Beilhack - Nelita Du Plessis - Gerhard Walzl( - Ulrike Schleicher - Uwe Ritter - Manfred B. Lutz ......................................................................................... 52

P31 DENDRITIC CELLS PRE-CONDITION NAIVE T CELLS TO PROMOTE THEIR EFFECTOR DIFFERENTIATION POTENTIAL

Thomas Kreutzberg - Caroline Wrangler - Christine Schmitt-Mbamunyo - Günter J. Hämmerling - Natalio Garbi ...................................................................................................................................... 53

P32 RELMα EXPRESSING MACROPHAGES PROVIDE SKIN BARRIER FUNCTION AND PROTECT FROM FATAL LUNG DAMAGE DURING HELMINTH INFECTION

Branislav Krljanac - Christoph Koch - Roland Naumann - Stefan Wirtz - Stephan Culemann - Gerhard Krönke - David Voehringer ................................................................................................................. 54




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P33 COMBINED PHOSPHOPROTEOME AND TRANSCRIPTOME ANALYSIS OF MACROPHAGES STIMULATED WITH MYCOBACTERIAL CORD FACTOR REVEALS A DICHOTOMY OF MINCLE-DEPENDENT AND -INDEPENDENT SIGNALING

Roland Lang - Madlen Hansen - Julian Peltier - Barbara Killy - Gernot Schabbauer ...................... 55

P34 CELL-TYPE SPECIFIC EXPRESSION OF HFE DETERMINES THE COURSE OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM INFECTION IN MICE

Christoph Metzendorf - Maja Vujic-spasic( - Andrea Schroll - David Haschka - Alexander Hoffmann - Richard Sparla - Christian Huck - Heribert Talasz - Matthias Hentze - Martina Muckenthaler - Günter Weiss - Manfred Nairz ........................................................................................................................ 56

P35 NA+ BOOSTS ANTIBACTERIAL DEFENSE BY ENHANCING NFAT5-DEPENDENT AUTOPHAGOLYSOSOME FORMATION

Patrick Neubert - Andrea Weichselbaum - Carmen Reitinger - Valentin Schatz - Agnes Schröder - John R. Ferdinand - Anna-Lorena Bär - Christoph Brochhausen - Roman G. Gerlach - Stefan Tomiuk - Ger Van Zandbergen - Katrina Binger - Dominik N. Müller - Kento Kitada - Menna R. Clatworthy - Christian Kurts - Zeinab Abdullah - Jonathan Jantsch ..................................................................... 57

P37 MYELOID PTEN PROMOTES OBESITY-INDUCED INSULIN RESISTANCE Julia Stefanie Brunner - Andrea Vogel - Alexander Lercher - Mario Kuttke - Omar Sharif - Gernot

Schabbauer .......................................................................................................................................... 58

P38 INFLAMMATORY MONOCYTE-DERIVED MACROPHAGE WNT SIGNALLING REQUIRED FOR BRAIN REGENERATION

Claire Davies - Anna Williams - Veronique Miron ............................................................................ 59

P39 A UNIQUE MACROPHAGE SUBSET THAT PLAYS A ROLE IN SARCOID GRANULOMA FORMATION Clarice Lim - Stefanie Hörer - Julia Matschinger - Karine Köhrer - Thomas Krausgruber - Thomas

Weichhart ............................................................................................................................................ 60

P40 IMMUNOSUPPRESSIVE BONE-MARROW DERIVED MACROPHAGES ACCUMULATE IN THE CORE OF HUMAN GLIOBLASTOMAS

Laura Pinton - Elena Masetto - Marina Vettore - Samantha Solito - Sara Magri - Marta D’andolfi - Paola Del Bianco - Giovanna Lollo - Alessandro Della Puppa - Susanna Mandruzzato ................. 61

P41 DENDRITIC CELL PRODUCTION OF THE CHEMOKINE CCL17 IS REQUIRED FOR OPTIMAL TH2 RESPONSE INDUCTION AGAINST BOTH HELMINTHS AND ALLERGENS

Amanda Ridley - A.T. Phythian Adams - F.R. Svedberg - D. Cunoosamy - I. Förster - E.M. Bignell - M. Feeney - P.C. Cook - A.S. Macdonald ................................................................................................. 62

P42 MIR-146A A KEY PLAYER IN BONE METABOLISM Victoria Saferding - Melanie Hofmann - Julia S Brunner - Mihaela F. Militaru - Antonia Puchner -

Silvia Hayer - Gernot Schabbauer - Melanie Timmen - Richard Stange - Josef S . Smolen - Stephan Blueml .................................................................................................................................................. 63

P43 NA+ INDUCED NFAT5 AS A NOVEL REGULATOR OF OSTEOCLASTOGENESIS Agnes Schröder - Patrick Neubert - Jens Titze - Aline Bozec - Wolfgang Neuhofer - Peter Proff - Christian

Kirschneck - Jonathan Jantsch ............................................................................................................. 64

P44 SERA OF PATIENTS WITH AXIAL SPONDYLOARTHRITIS (AXSPA) ENHANCE OSTEOCLASTOGENIC POTENTIAL OF MONOCYTES ISOLATED FROM HEALTHY INDIVIDUALS

Mariusz Korkosz - Marcin Czepiel - Zofia Gula - Malgorzata Stec - Kazimierz Weglarczyk - Magdalena Rutkowska-zapala - Anna Gruca - Marzena Lenart - Jaroslaw Baran - Jerzy Gasowski - Przemyslaw Blyszczuk - Maciej Siedlar ................................................................................................................... 65




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P45 CHROMATIN STATE DYNAMICS AND GENE REGULATION IN MACROPHAGES DURING SKELETAL MUSCLE REGENERATION UPON AN ACUTE STERILE INJURY

Petros Tzerpos - Andreas Patsalos - Gergely Nagy - Nikolas Giannakis - Laszlo Holasz - Balazs Dezso- Laszlo Nagy ......................................................................................................................................... 66

P46 LUNG DENDRITIC CELLS AS KEY MEDIATORS LINKING CIGARETTE SMOKE AND LUNG DISEASE DEVELOPMENT IN RHEUMATOID ARTHRITIS

Robert Vassallo .................................................................................................................................... 67

P47 THE ROLE OF PI3K/PTEN IN MICROGLIA FUNCTIONS DURING HOMEOSTASIS AND NEUROINFLAMMATION

Andrea Vogel - Melanie Hofmann - Julia S. Brunner - Martina Kerndl - Hannes Datler - Omar Sharif - Gernot Schabbauer ............................................................................................................................. 68

P48 AN INTEGRATIVE ANALYSIS OF TRANSCRIPTOMICS AND LIPIDOMICS DATA FROM HUMAN CD14+ MONOCYTES

Ioanna Gemünd - Marie Oestreich - Lea Seep - Anna-lena Hardt - Thomas Ulas - Christoph Thiele - Joachim L. Schultze - Anna C. Aschenbrenner................................................................................... 69

P49 TOLERANCE INDUCED BY IL-35IG-EXPRESSING DENDRITIC CELLS IS MEDIATED BY ARGINASE 1 Maria Laura Belladonna - Eleonora Panfili - Giada Mondanelli - Claudia Volpi - Ciriana Orabona -

Ursula Grohmann ............................................................................................................................... 70

P50 EFFECT OF THE AIR POLLUTION – TRANSITION METAL CONTAINING PARTICULATE MATTER ON THE ACTIVITY OF HUMAN MONOCYTES

Adrianna Galuszka - Malgorzata Stec - Anna Gruca - Maciej Siedlar - Jarek Baran ...................... 71

P51 A NOVEL KYNURENINE-DEPENDENT CIRCUIT IN DC1 PROMOTE IDO1 EXPRESSION IN DC2 LEADING TO EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS SUPPRESSION

Marco Gargaro - Giulia Scalisi - Carlos G. Briseño - Giorgia Manni - Vivek Durai - Prachi Bagadia - Derek J. Theisen - Dario Nardi - Paolo Puccetti - Theresa L. Murphy - Kenneth M. Murphy - Francesca Fallarino .............................................................................................................................................. 72

P52 INDOLEAMINE 2,3-DIOXYGENASE 1 (IDO1): RELATIONSHIP BETWEEN FUNCTIONS AND INTRACELLULAR LOCALIZATION IN PLASMACYTOID DENDRITIC CELLS

Alberta Iacono - Andrea Pompa - Francesca De Marchis - Michele Bellucci - Fabio Grassi - Elisa Albini

- Ursula Grohmann - Maria Teresa Pallotta ...................................................................................... 73

P53 HYPERGLYCAEMIC CONTROL OF HISTONE CODE IN METABOLIC INFLAMMATION Marije Mossel - Kondaiah Moganti - Harald Klüter - Martin Harmsen - Julia Kzhyshkowska ....... 74

P54 N-ACETYLSEROTONIN RESTRAINS NEUROINFLAMMATION IN AN IDO1-MEDIATED FASHION IN DCS Eleonora Panfili - Alessandra Scarponi - Claudia Volpi - Antonio Macchiarulo - Laura Santambrogio -

Ursula Grohmann ............................................................................................................................... 75

P55 MYELOID DYSFUNCTION IN AN INDUCIBLE MOUSE MODEL OF GLYCOGEN STORAGE DISEASE TYPE 1B Federica Raggi - Anna Livia Pissavino - Roberta Resaz - Daniela Segalerba - Andrea Puglisi - Francesca

Antonini - Genny Del Zotto - Alessandra Gamberucci - Paola Marcolongo - Luca Mastracci - Federica Grillo - Maria Carla Bosco - Luigi Varesio - Alessandra Eva ........................................................... 76

P56 THE IMPACT OF THE PGD2-DP1-DP2 AXIS IN RHEUMATOID ARTHRITIS David Roula - Kathrin Rohrer - Martin Stradner - Eva Sturm - Akos Heinemann .......................... 77




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P57 N-ACETYLSEROTONIN EXERTS NEUROPROTECTIVE EFFECTS ENHANCING IDO1 CATALYTIC ACTIVITY Alessandra Scarponi - Eleonora Panfili - Claudia Volpi - Antonio Macchiarulo - Laura Santambrogio -

Ursula Grohmann ............................................................................................................................... 78

P58 HYPERGLYCEMIA MODULATES INFLAMMATORY RESPONSES TO METAL NANOPARTICLES IN HUMAN MACROPHAGES

Tatjana Sevastyanova - Alexandru Gudima - Vladimir Ryabov - Nihal Engin Vrana - Harald Klüter - Julia Khzyshkowska ............................................................................................................................ 79

P59 NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT) ACTS AS A METABOLIC GATE OF SUPPRESSOR MYELOID CELLS MOBILIZATION

Cristina Travelli - Sabina Sangaletti - Ubaldina Galli - Mario Paolo Colombo - Armando A. Genazzani

- Maria Francesca Consonni - Antonio Sica ...................................................................................... 80

P60 RESOLUTION OF CUTANEOUS LEISHMANIASIS AND PERSISTENCE OF LEISHMANIA MAJOR IN THE ABSENCE OF ARGINASE 1

Katrin Paduch - Andrea Debus - Ulrike Schleicher - Christian Bogdan .......................................... 81

P61 HIF-PROLYLYDROXYLASE-2 (PHD2) IS A FUNDAMENTAL REGULATOR OF NEUTROPHIL MIGRATION IN INNATE IMMUNITY

Sundary Sormendi - Ana Meneses - Iannis Kourtzelis - Pablo Saez - Pablo Vargas - Triantafyllos Chavakis - Ben Wielockx .................................................................................................................... 82

P62 THERAPEUTIC OPTIONS FROM THE UNDERSTANDING OF MACROPHAGE HETEROGENEITY IN THE TUMOR MICROENVIRONMENT

B. Savino - E. Setten - I. Mattiola - D. Mavilio - M. Locati .............................................................. 83

P63 PHENOTYPIC AND TRANSCRIPTIONAL PROFILING REVEALS MONOCYTE-RELATED MACROPHAGES PHENOTYPICALLY RESEMBLING DC IN HUMAN INTESTINE

L. Richter - O. J. B. Landsverk - N. Atlasy - A. Bujko - S. Yaqub - R. Horneland - O. Øyen - E. M. Aandahl - K. E. A. Lundin - H. G. Stunnenberg - E. S. Bækkevold - F. L. Jahnsen ......................... 84




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OC1/1SMALL MOLECULE MODULATORS OF PRION PROTEIN (PRPC) IN DENDRITIC CELLS REGULATE INFLAMMATORY RESPONSES IN A MODEL OF MULTIPLE SCLEROSIS

Giorgia Manni(1) - M. Letizia Barreca(2) - Giulia Scalisi(1) - Emiliano Basini(3) - Marco Gargaro(1) - Giuseppe Manfroni(2) - Francesca Fallarino(1)

University of Perugia, Department of Experimental Medicine, Perugia, Italy(1) - University of Perugia, Department of Pharmaceutical Sciences, Perugia, Italy(2) - University of Trento, Dulbecco Telethon Laboratory of Prions & Amyloids, Center for Integrative Biology (CIBIO), Trento, Italy (3)

Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for human multiple sclerosis, an autoimmune disease driven by differentiated Th1 and Th17 cells, and inflammatory dendritic cells (DCs). Recent data show that EAE is worsened in mice lacking PrPC protein and disease exacerbation has been attributed to T cells, which would differentiate into more aggressive effectors, when deprived of PrPC. Novel compounds, modulating PrPC activity, have been recently developed, although not tested in EAE models yet. Interestingly, by specific gene array studies, we found that PrPC was highly expressed in DCs and differentially expressed among selected DC subsets. Moreover, by using a series of computational, and biochemical assays we identified novel small molecules, able to modulate PrPC function in specific cell lines overexpressing PrPC. By employing such tools, we studied the impact of PrPC modulation in promoting regulatory DC subsets. To this aim, DCs were treated either with novel PrPC modulators or a reference PrPC binding molecule Fe (III)-TMPyP. Then, they were co-cultured with sorted naïve CD4+ T cells in vitro. Notably, we found a significant expansion of FOXP3+CD4+ T regulatory (Treg) cells in cultures containing DC subsets, which were pre-treated with the specific PrPC modulators. Moreover, in a EAE model, we showed that systemic administration of such PrPC regulators, resulted in significant reduction of disease severity, compared to untreated controls. Overall, our results suggest that PrPC modulation in selected DC subsets may represent a novel means to restrain inflammatory T cell effectors, resulting in reduced inflammation, demyelination, and axonal injury in a model of EAE.

1 · MYELOID CELL SUBSETS




EMDS2018

12

OC2/1NEUTROPHIL TRAPS IN LEUKEMIA: FROM TRIGGERS OF DISEASE PROGRESSION TO VEHICLE FOR NEW VACCINES

Sabina Sangaletti(1) - Claudio Tripodo(2) - Mario Paolo Colombo(1)

Fondazione Irccs Istituto Nazionale Dei Tumori, Research Department, Milan(1) - University of Palermo, Department of Health Sciences, Palermo(2)

Neutrophil extracellular traps (NET) are DNA threads decorated with anti-microbial proteins released by neutrophils to control microbial infections. NET receive increasing attention because of their pathogenic involvement in autoimmunity and in lymphomagenesis. In such a context, we have demonstrated a role of NET in the malignant transformation of B1 cells in autoimmunity-prone Fas mutant mice stably knocked down for the stromal master regulator SPARC. Epidemiological studies have shown that infections and autoimmunity increase the risk of myeloid malignancies but mechanisms are still unknown. Mutations in the nucleolar protein nucleophosmin (NPM) are among the most frequent molecular alterations in AML and are an important prognostic marker. NPM1 regulates ARF-p53 tumor-suppressor pathway. Its mutations cause the relocation of the protein from nucleous to cytoplasm (NPMc) that, per se, is sufficient to trigger an AML. AML patients with NPM1 mutation show improved overall survival because of a T-cell response against the mutated epitopes. This suggests that the intrinsic immunogenicity of mutated NPM1 could be amplified by presentation on the NET as for the cytoplasmic antimicrobial proteins. In this context neutrophils can be harnessed to produce NET delivering mutated NMP1 as effective vehicles for NPM1c vaccination, a new strategy for treating minimal residual disease or to prolong maintenance of disease free condition. By using NPMc transgenic mice that express the human mutated nucleophosmin (NPMc) and develop a mild myeloproliferation we demonstrated that NET represent the pathogenic link between autoimmunity and myeloid malignancies and, at the same time, could be a vehicle for vaccination against leukemic cell-associated antigens. We also demonstrated that the down-modulation of the ECM protein SPARC, which occurs as part of the early BM stromal changes associated with pre-leukemic state, could favor loss of tolerance and NET formation in BM undergoing a myeloproliferative spur. Besides the role of NET in triggering the transition from autoimmunity to leukemia, we investigated the hypothesis that DC-loaded with NET could be used as a vaccine against leukemia-associated antigens, such as the prototypical NPMc mutant. Mutant NPMc marks the cytoplasm of immature cells within the BM of NPMc transgenic mice, but not their mature progeny. Thus, the effective immunization using NPMc-derived NET should allow selective elimination of pre-leukemic NPMc+ progenitor cells but not that of residual normal progenitors or mature myeloid cells.





EMDS2018

13

OC3/2IL23 SECRETED BY MYELOID CELLS DRIVES CASTRATION RESISTANT PROSTATE CANCER

Arianna Calcinotto(1) - Clarissa Spataro(1) - Elena Zagato(1) - Johann De Bono(2) - Andrea Alimonti(3)

Institute of Oncology Research, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland(1) - The Institute of Cancer Research and The Royal Marsden Nhs Foundation Trust, The Institute of Cancer Research and The Royal Marsden Nhs Foundation Trust, London, United Kingdom(2) - Institute of Oncology Research, Università della Svizzera Italiana, Bellinzona, Switzerland(3)

Prostate cancer causes more than 300,000 deaths annually worldwide and is one of the most common causes of cancer-linked mortality in men. Prostate-cancer treatment usually fails after time as resistance to therapy develops, a condition known as castration-resistant prostate cancer (CRPC). Acquiring a better understanding of the mechanisms that control the development of CRPC remains an unmet clinical need. The well-established dependency of cancer cells on the tumour microenvironment indicates that the microenvironment might control the emergence of CRPC. We recently identify that IL-23 produced by myeloid-derived suppressor cells (MDSCs) is the driver of treatment resistance in mice and patients with CRPC. Mechanistically, IL-23 secreted by MDSCs can activate the androgen receptor pathway in prostate tumour cells, promoting cell survival and proliferation in androgen-deprived conditions. Intra-tumour MDSC infiltration and IL-23 concentration are increased in blood and tumour samples from patients with CRPC. Antibody-mediated inactivation of IL-23 restored sensitivity to androgen-deprivation therapy in mice. Furthermore, the use of pharmacological techniques to deplete MDSCs delayed the emergence of castration resistance in three different mouse models. Taken together, these results reveal that MDSCs promote CRPC by acting in a non-cell autonomous manner. Treatments that block IL-23 can oppose MDSC-mediated resistance to castration in prostate cancer and synergize with standard therapies. This discovery could have important clinical implications and advances our understanding of the biological processes that underlie castration resistance.

2 · CANCER




EMDS2018

14

OC4/2CCR1 AND CCR5 CONTROL THE POLARIZATION COMMITMENT OF EARLY MYELOID PRECURSORS IN TUMOR HOST

Serena Zilio(1) - Donald T. Weed(2) - Alessia Zoso(3) - Dimitri Van Simaeys(1) - Carla Rodriguez(4) - Emilia Mazza(5) - Silvio Bicciato(5) - Vincenzo Bronte(6) - Paolo Serafini(1)

University of Miami, Microbiology and Immunology Department, Miami, United States(1) - University of Miami, Department of Otolaryngology, Miami, United States(2) - University of Miami, Diabetes Research Institute, Miami, United States(3) - University of Miami, Microbiology and Immunology Department, Miami, United States(4) - Univeristy of Modena and Reggio Emilia, Life Sciences Department, Modena, Italy(5) - Verona University Hospital, Department of Medicine, Verona, Italy(6)

Tumors produce growth factors and cytokines that are sufficient to induce the differentiation of bone marrow (BM) cells into myeloid derived suppressor cells (MDSCs) and “type 2” myeloid cells that provide immune protection, stimulate neoplastic cell growth, invasiveness, and metastasis, and impair the efficacy of conventional and experimental therapies. By analyzing the first differentiation steps of BM cell cultures in the presence of tumor conditioned media we discovered that tumor derived factors secreted from different mouse tumors induce BM cells to produce and secrete CCL3 and CCL4 that autocrinally promote MDSC differentiation. The simultaneous blockade of CCL3 and CCL4 cognate receptors CCR1 and CCR5 during BM cell differentiation caused profound changes in myeloid cell composition. In particular, in vitro inhibition of CCR1/5 inhibits MDSC differentiation and allows the generation of tumoricidal neutrophils and dendritic cells. To better understand the role of CCR1 and CCR5 during MDSC differentiation, we used the 4PD nanoparticles that allow the selective targeting of IL4Rα-expressing MDSCs and myeloid progenitors in vivo: CCR1/5 silencing significantly modified the tumor microenvironment composition and restrained tumor growth. This did not derive from an impaired chemotaxis of MDSCs into the tumor but rather from the repolarization of both macrophages and dendritic cells toward a type 1 anti-tumoral phenotype with strong tumoricidal activity.The key role of CCR1 and CCR5 in myeloid cell polarization was found true in humans: CCR1/5 blockade inhibits MDSC differentiation from hematopoietic stem and progenitor cells isolated either from umbilical cord blood or from the blood of patients with head and neck cancer. Our findings indicate that CCR1 and CCR5 are an important immunological switch that control the polarization commitment of myeloid cells and suggest that targeted inhibition of CCR1/5 on MDSCs and myeloid progenitors can be a new and effective anti-tumor immune-therapeutic strategy to convert pro-tumoral MDSCs into tumoricidal myeloid cells.

2 · CANCER




EMDS2018

15

OC5/3GLUCOCORTICOIDS IMPAIR PHAGOCYTOSIS AND INFLAMMATORY RESPONSE IN A MODEL OF HUMAN MACROPHAGES

Mauricio Olivares-Morales(1), Marjorie De la Fuente(1), Karen Dubois(1), Daniela Parada(1), David Diaz-Jiménez(1), Xiaojiang Xu(2), Nayaret Chamorro(3) Rodrigo Quera(6), John Cidlowski(7), Roberto Vidal(3), Marcela A. Hermoso(1)

Innate Immunity Laboratory, Immunology program, ICBM, Faculty of Medicine, Universidad de Chile. Santiago, Chile(1) - Laboratory of Integrative Bioinformatics, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, NC, USA(2) - Enteropathogens Laboratory, Microbiology and Mycology Program, Biomedical Sciences Institute, Faculty of Medicine, Universidad de Chile. Santiago, Chile(3)- Neuroimmunology Laboratory, Immunology program, Biomedical Sciences Institute, Faculty of Medicine, Universidad de Chile. Santiago, Chile(4) - Cell Biology Program, Biomedical Sciences Institute, Faculty of Medicine, Universidad de Chile. Santiago, Chile(5) - Gastroenterology Department, Clínica Las Condes, Santiago, Chile(6) - Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, NC, USA(7)

Glucocorticoids are the first line treatment for Crohn’s disease and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages. Crohn’s disease is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors, such as adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. Additionally, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Lastly, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that after bacterial infection Glucocorticoids impair phagocytosis and induce anti-inflammatory polarization, possibly contributing to the survival of AIEC in infected Crohn’s disease patients.FONDECYT 1170648

3 · INFECTIONS




EMDS2018

16

OC6/3CD16+ MONOCYTES ARE PRECURSORS FOR DENDRITIC CELLS WITH RALDH ACTIVITY THAT PROMOTE PATHOGENICITY DURING HIV-1 INFECTION.

Amélie Cattin(1) - Vanessa Sue Wacleche(1) - Natalia Fonseca Do Rosario(2) - Jean-Philippe Goulet(3) - Dominique Gauchat(2) - Annie Gosselin(2) - Jean-Pierre Routy(4) - Petronela Ancuta(1)

Chum Research Centre, Department of Microbiology, Infectiology, and Immunology, Faculty of Medicine, Université De Montréal, Montreal, Canada(1) - Chum Research Centre, Chum Research Centre, Montreal, Canada(2) - Caprion, Caprion, Montreal, Canada(3) - Mcgill University Health Centre, Chronic Viral Illness Service and Division of Hematology, Mcgill University, Montreal, Canada(4)

The human immunodeficiency virus type 1 (HIV) exploits key features of the immune system for its dissemination and persistence. The expansion of intermediate/non-classical CD16+ monocytes (Mo) represents a hallmark of HIV infection. Given the fact that Mo are an important pool of dendritic cell (DC) precursors, we investigated differences in the ability of CD16+ versus CD16- Mo-derived DC (MDDC) to present antigens and disseminate HIV to CD4+T-cells and explored molecular mechanisms underlying these differences.FACS-sorted monocytes were differentiated into MDDC. Genome-wide transcriptional profiling was performed using the Affymetrix technology. The immunogenic and trans HIV infection potential of MDDC was evaluated by FACS upon exposure to SEB, CMV, and S. aureus and co-culture with autologous CD4+T-cells.CD16+MDDC distinguished from CD16-MDDC by a superior ability to transmit HIV to CD4+T-cells proliferating in response to S. aureus. This coincided with high CCR5 and integrin β7 expression on S. aureus-specific CD4+T-cells. Despite similar expression of classical DC markers, transcriptional profiling and RT-PCR identified sets of transcripts preferentially expressed in CD16+MDDC versus CD16-MDDC, including the integrin αE/CD103, the retinoic acid (RA)-synthesizing enzyme RALDH and the transcription factor TCF4. RALDH activity was higher in CD16+MDDC versus CD16-MDDC and was induced by S. aureus. of note, the MDDC-mediated HIV trans-infection of CD4+T-cells was diminished in the presence of the RA-receptor antagonist LE540. Finally, the inhibition of the TCF4/β-catenin pathway led to decreased RALDH activity and limited HIV trans infection.Thus, CD16+ Mo versus CD16- Mo are precursors for CD103+DC with a unique ability to metabolize vitamin A into RA, a RALDH-dependent metabolic pathway under the transcriptional control of TCF4 and hijacked by HIV for efficient DC-to-T-cell transmission. These results point to RALDH as an important determinant of CD16+MDDC pathogenicity during HIV transmission at mucosal levels, as well as a contributor to HIV reservoir persistence during antiretroviral therapy.

3 · INFECTIONS




EMDS2018

17

OC7/4

MAPPING THE HUMAN IMMUNE LANDSCAPE IN THE ALVEOLAR SPACE OF HEALTHY DONORS AND COPD PATIENTS

Kevin Baßler(1) - Wataru Fujii(2) - Theodoros Kapellos(3) - Carmen Pizarro(4) - Drik Skowasch(5) - Joachim Schultze(6)

Life and Medical Sciences Institute Bonn, University of Bonn, Bonn, Germany(1) - Life and Medical Sciences Bonn, University of Bonn, Bonn, Germany(2) - Life and Medical Sciences Bonn, University Bonn, Bonn, Germany(3) - Medical Clinics and Policlinics, University Hospital Bonn, Bonn, Germany(4) - Medical Clinics and Policlinics, University of Bonn, Bonn, Germany(5) - Platform for Single Cell Genomics and Epigenomics At The German Center for Neurodegenerative Diseases, Dzne, Bonn, Germany(6)

Chronic obstructive pulmonary disease (COPD) is ranked the fourth leading cause of death worldwide and hence constitutes a significant burden, both medically and financially. The disease is characterized by a poorly reversible airway obstruction which is mainly caused by chronic inflammation of the lung followed by destruction of the parenchyma. However, the cellular mechanism underlying COPD is poorly understood and thus no effective therapy is currently available. To broaden the current knowledge of the immune landscape in the lung, we performed single cell RNA-Seq of CD45-enriched bronchoalveolar lavage cells obtained from five COPD patients and five healthy controls. In all donors, we found that alveolar macrophages (AMs) constitutes the major immune cell population and together with mast cells, B cells, T cells and dendritic cells, they form the basic immune compartment in the alveolar space. Interestingly, we detected an enrichment of granulocytes in COPD patients. We confirmed this finding by a large-scale multi-color flow cytometry study, which comprised 26 COPD patients and 20 healthy controls. Further analysis revealed sub-populations within the AM compartment. To our knowledge, these sub-populations have not been described before in the alveolar space and we classified them into proliferating BIRC5+ AMs, immature AMs, mature AMs and monocyte-derived cells. Using diffusion maps, we were able to model a developmental trajectory, which indicates that AM are replenished in at least two different ways, namely via proliferating AMs (self-replenishment) and differentiating monocytes.Taken together the current single cell RNA-Seq data allows insight into the immune landscape of the alveolar space, which has never been provided in such a detail before. Moreover, the inclusion of COPD samples provides a platform to study the pathogenesis of the disease and might help to unravel the underlying cellular mechanisms.

4 · SINGLE CELL ANALYSIS OF MYELOID CELLS




EMDS2018

18

OC8/4INTEGRATION OF IMMUNE STIMULI AT THE SINGLE-CELL LEVEL EXPANDS THE SPECTRUM OF MACROPHAGE ACTIVATION STATES AND IDENTIFIES TARGETS FOR FUNCTIONAL REPROGRAMMING

Marco Genua(1) - Francesco Cilenti(1) - Giulia Barbiera(1) - Dario Iodice(1) - Elisa Montaldo(1) - Eleonora Lusito(1) - Renato Ostuni(1)

San Raffaele - Telethon Institute for Gene Therapy (sr-tiget), Irccs San Raffaele Scientific Institute, Milan, Italy(1)

Macrophages are specialized cells capable of translating complex environmental signals into specific and partially reversible activation states. Yet, while the genomic features of responses to individual immune stimuli are well characterized, it remains unclear how complex inputs, entailing multiple signals with distinct biological properties, are computed and translated into transcriptional outputs. We have recently showed that co-exposure of bone marrow-derived macrophages (BMDMs) to the paradigmatic cytokines IFN-gamma and IL-4 leads to broad transcriptional cross-antagonisms driven by specific genomic features of co-targeted gene regulatory elements (Piccolo et al., Nature Immunology 2017). Here, we expand these studies using bulk and single-cell genomics to show that macrophages are intrinsically able to integrate relevant combinations of immune stimulatory and suppressive signals into coherent gene expression programs. This type of response was globally homogeneous, generalizable across multiple combinations of stimuli, and resulted in the acquisition of unique and emerging transcriptional features distinct from those of cells exposed to individual stimuli. Mechanistic analyses revealed common principles underlying selectivity and specificity of transcriptional cross-antagonisms, which we incorporated into gene therapy approaches for functional macrophage reprogramming in cancer.Our analyses expand the spectrum of macrophage activation states by investigating a novel dimension, namely integration of antagonistic stimuli into emerging and unique transcriptional programs. Since macrophages, in vivo, are invariably exposed to multiple stimuli at the same time, we argue that these data will help to better interpret macrophage polarization in homeostasis and disease.





EMDS2018

19

OC9/4SINGLE-CELL CHARACTERIZATION OF ADIPOSE TISSUE MACROPHAGES

André Sulen(1) - Emelie Barreby(1) - Francisco Verdeguer(2) - Camille Bleriot(3) - Florent Ginhoux(3) - Myriam Aouadi(1)

Integrated Cardio Metabolic Centre, Karolinska Instituet / Department of Medicine Huddinge, Stockholm, Sweden(1) - Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland(2) - Singapore Immunology Network (SIGN), Agency For Science, Technology and Research (A(*)STAR), Singapore, Singapore(3)

Macrophages are important players in obesity-associated insulin resistance and Type 2 Diabetes. In adipose tissue, macrophages secrete inflammatory cytokines that inhibit the action of insulin, this leads to detrimental hyperglycemia. Conversely, macrophages are also required for maintenance of adipose tissue homeostasis by regulating angiogenesis, extracellular matrix remodeling, lipid storage and clearance of dead cells. Therefore, macrophages within this tissue play both beneficial and detrimental roles in insulin sensitivity. Although extensive studies have described macrophage properties in adipose tissue, their phenotypic heterogeneity and its regulation remains poorly described on the single-cell level. This project aims to define adipose macrophage heterogeneity with single-cell methodologies, during both lean and obese conditions in both mice and humans. Visceral adipose tissue stromal vascular fractions (SVF) from lean and obese mice were analyzed by mass cytometry (CyTOF) and single-cell RNA sequencing (sc-RNA-seq). CyTOF analysis was carried out with a panel of 40 myeloid-relevant markers. The scRNA-seq was carried out with the Smart-seq2 protocol optimized for adipose macrophages. Mass cytometric analysis of SVF readily identified Adipocyte Progenitors, Endothelial cells, T-cells, B-cells, Neutrophils, Eosinophils and several populations of CD68+ myeloid cells, all expected cell types in adipose tissue. Relevant to metabolic disease, the well-described macrophage phenotypic shift from CD206+CD11c- to CD206-CD11c+ cells during obesity was observed. Further, single-cell RNA-seq analysis show the emergence of a macrophage population with increased expression of genes involved in lipid-handling (Plin2, Lpl), benchmarking these datasets in the context of metabolic disease. This unbiased single-cell characterization of adipose tissue immune cells, on both protein and transcription level, provides an improved phenotypic description of immune cells in adipose tissue. This will ultimately be used to identify novel mechanisms for regulation of adipose macrophages and their effects on metabolism.





EMDS2018

20

OC10/5MTOR SIGNALING IN MACROPHAGES CONTRIBUTES TO THE MAINTENANCE OF IRON HOMEOSTASIS

Nyamdelger Sukhbaatar(1) - Monika Linke(1) - Maria Schöller(1) - Igor Theurl(2) - Günter Weiss(2) - Stephanie Deborah Fritsch(1) - Stefanie Horer(1) - Barbara Scheiber-Mojdehkar(3) - Markus Hengstschläger(1) - Thomas Weichhart(1)

Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria(1) - Department of Internal Medicine Vi, Infectious Diseases, Immunology, Rheumatology, Pneumology, Medical University of Innsbruck, Innsbruck, Austria(2) - Institute of Medical Chemistry and Pathobiochemistry, Medical University of Vienna, Vienna, Austria(3)

Macrophages are essential for maintaining iron homeostasis and erythropoiesis. However, the molecular pathways that regulate iron homeostasis in macrophages are ill-defined. Therefore, we assessed a potential role of mTORC1 for iron recycling in macrophages, in which macrophage-specific mTORC1 hyperactivation is induced by deletion of its negative regulator TSC2 (TSC2ΔM mice).Freshly isolated bone marrow (BM) from TSC2ΔM mice was pale suggesting a defect in erythropoiesis. In line, reduced numbers of both immature and mature erythrocytes as well as significant lower numbers of erythroblastic islands in BM from TSC2ΔM mice indicated a failure in steady state erythropoiesis. In contrast, an enhanced stress erythropoiesis in the spleen of TSC2ΔM mice was observed, which balanced systemic erythrocyte levels. Nevertheless, red blood cells from TSC2ΔM mice were microcytic, hypochromic and had a significantly prolonged half-life. Moreover, a highly reduced transferrin saturation in serum and a lack of non-heme iron stores in the BM and spleen, as well as strongly diminished hepcidin expression in the liver of the TSC2ΔM mice were indicative of a disturbed iron metabolism. Interestingly, inhibition of mTORC1 with everolimus completely restored erythropoiesis in the BM of TSC2ΔM mice as well as iron storage. These results suggested that the erythropoiesis defect is caused by a dysfunctional regulation of iron by macrophages and that mTORC1 in macrophages is critical in maintaining iron homeostasis and steady-state erythropoiesis. The mTORC1-dependent molecular mechanisms that control cellular iron metabolism in the macrophages are being further investigated.

5 · MYELOID CELLS IN TISSUES




EMDS2018

21

OC11/5DYNAMIC AGE-DEPENDENT CHANGES IN THE DEVELOPMENTAL ORIGIN OF MONONUCLEAR PHAGOCYTES IN THE KIDNEY

Natallia Salei(1) - Stephan Rambichler(1) - Nikolaos Papaioannou(2) - Johanna Salvermoser(1) - Ronja Schuchert(3) - Filippo Cernilogar(4) - Gunnar Schotta(4) - Christian Schulz(3) - Barbara U. Schraml(1)

Walter-brendel-centre For Experimental Medicine, University Hospital, Lmu, Munich, Germany(1) - Biomedical Center, Ludwig Maximilian University, Munich, Germany(2) - Medizinische Klinik Und Poliklinik I, Klinikum Der Universität, Lmu, Munich, Germany(3) - Biomedical Center and Center For Integrated Protein Science Munich, Ludwig Maximilian University, Planegg-martinsried, Germany(4)

The kidney contains a dense network of mononuclear phagocytes; yet, relatively little is known about the origin and functions of specific subsets of mononuclear phagocytes in this organ. In adult mice, the kidney harbors conventional dendritic cells of the cDC1 and cDC2 subtypes, as well as a fraction of CD64+ cells that can be divided into CD11bhighF4/80low cells and CD11blowF4/80high cells, both phenotypically strongly resembling macrophages. Using fate mapping we reveal that the CD64+ cells in kidney are ontogenetically diverse and develop from distinct hematopoietic progenitors throughout life. CD11bhighF4/80low cells arise from definitive hematopoiesis shortly after birth, resemble cDC2 in their developmental requirements but show dendritic cell origin first in early adulthood. In contrast, CD11blowF4/80high cells arise from primitive yolk sac progenitors during embryogenesis. By 4 weeks of age these yolk sac derived macrophages are replaced by a population of CD11blowF4/80high cells arising from definitive hematopoiesis. These cells are initially established independent of dendritic cell progenitors but in adulthood CD11blowF4/80high cells arise almost exclusively from conventional dendritic cell progenitors. Despite their dendritic cell origin in adulthood, CD11blowF4/80high cells are long-lived and maintain a macrophage-specific gene signature. Our observations identify distinct developmental pathways for two different mononuclear phagocyte populations in kidney and have important implications for the further dissection of their distinct roles in organ homeostasis and immunity.





EMDS2018

22

OC12/6IL10 POLARIZATION REGULATES TNF-SIGNALING AND MITOCHONDRIAL FUNCTION IN HUMAN MACROPHAGES

Hung-Jen Chen(1) - Guillermo Griffith(1) - Menno De Winther(1)

Amsterdam Umc (AMC), Medical Biochemistry, Amsterdam, Netherlands(1)

Interleukin 10 (IL10) is an anti-inflammatory cytokine regulating immune responses. The defect in either IL10 or its receptor (IL10R) has been associated with autoimmune diseases such as inflammatory bowel diseases (IBDs). However, the molecular basis of IL10’s anti-inflammatory features in human macrophages remains unclear.Using RNAseq and cytokine assays we show that IL10 polarization inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression and secretion in human monocyte-derived macrophages (MDMs). We clustered the transcriptomic data into eight gene sets with distinct expression patterns. These gene sets are enriched for specific pathways such as TNF-signaling, neutrophil activation, endocytosis and mitochondrial function.Interestingly, from these gene sets we observed decreased mitochondrion pathways and up-regulated mTORC1 signaling with IL10 polarization. This transcriptomic observation correlates with an increase of lactate secretion after IL10 priming of human macrophages, which has been shown the opposite in mouse macrophages treated with IL10 (Ip et al, Science, 2017).Next, we conducted gene set enrichment analysis (GSEA) of these 8 gene sets against publicly available IBD datasets. As expected, we found a suppression of mitochondrial gene sets and up-regulation of TNF-signaling gene clusters in IBD colon biopsies. Surprisingly, these 2 types of gene sets are all negatively enriched in the patient MDMs. One possible explanation is that the elevated seral IL10 in patients suppressed both TNF-signaling and mitochondria.We also performed GSEA on the colon biopsies from anti-TNF non-responders and responding Crohn’s disease patients (Schmitt et al. Gut 2018). Interestingly, only the gene sets correlating with mitochondria are negatively enriched in the non-responders. Moreover, we found that mitochondrion-related genes (GO:0005739) inhibited by IL10 after LPS stimulation gave the highest enrichment score, suggesting a correlation between anti-TNF non-responding IBDs and IL10-suppressed mitochondrial function.Taken together, we conclude that IL10 is a crucial regulator of TNF-signaling and the metabolism in human macrophages. Altering the metabolism affected by IL10 polarization while preserving its anti-inflammatory feature might serve as a new therapeutic direction for autoimmune diseases.

6 · IMMUNOMETABOLISM




EMDS2018

23

OC13/6SEMAPHORIN 6D REVERSE SIGNALING CONTROLS MACROPHAGE LIPID METABOLISM AND ANTI-INFLAMMATORY POLARIZATION

Sujin Kang(1) - Atsushi Kumanogoh(1)

Immunology Frontier Research Center, Osaka University, Immunopathology, Osaka, Japan(1)

Polarization of macrophages into pro-inflammatory or anti-inflammatory states has distinct metabolic requirements with mTOR kinase signaling playing a critical role. However, it remains unclear how mTOR regulates metabolic status to promote polarization of these cells. Here, we show that an mTOR–Semaphorin 6D (Sema6D)–Peroxisome proliferator receptor γ (PPARγ) axis plays critical roles in macrophage polarization. Inhibition of mTOR or loss of Sema6D blocked anti-inflammatory macrophage polarization, concomitant with severe impairments in PPARγ expression, uptake of fatty acids, and lipid metabolic reprogramming. Macrophage expression of the receptor Plexin-A4 is responsible for Sema6D-mediated anti-inflammatory polarization. We found that a tyrosine kinase, c-Abl, which associates with the cytoplasmic region of Sema6D, is required for PPARγ expression. Furthermore, Sema6D is important for generation of intestinal resident CX3CR1hi macrophages and prevent development of colitis. Collectively, these findings highlight crucial roles for Sema6D reverse signaling in macrophage polarization, coupling immunity and metabolism via PPARγ.





EMDS2018

24

P1ISOLATION AND CHARACTERIZATION OF FUNCTIONAL CROSS-PRESENTING DENDRITIC CELLS FROM MOUSE TISSUES AND TUMOR

Frank Ralph Morrissey-Wettey(1) - Cristina Conforti Andreoni(2) - Daniela Vorholt(1) - Andrzej Dzionek(3)

Miltenyi Biotec Gmbh, R&d Department, Bergisch Gladbach, Germany(1) - Miltenyi Biotec Gmbh, Marketing Department, Bergisch Gladbach, Germany(2) - Miltenyi Biotec Gmbh, R&d Department, Miltenyi Biotec Gmbh, Germany(3)

As sentinels of the immune system, dendritic cells (DCs) naturally populate several tissues, especially those located at the interface between the body and the external environment, such as skin, lung, and intestine. However, DCs are also present in lymphoid organs, where they activate T cells and thus start immune responses. Moreover, DCs are present in the tumor microenvironment, where they are involved in anti-tumor immune responses. In particular, the cDC1 subset expressing CLEC9A and XCR1 has the natural ability to cross-present exogenous antigens, thus playing a pivotal role in the induction of protective cytotoxic T lymphocyte responses, which are vital for the eradication of cancers and viral infections. In the past, studies of cross-presenting DCs were hampered by the cells’ low frequency, by the difficulty to obtain viable cells from tissues and by the lack of specific cell surface markers. Several approaches for the isolation of these cells are commonly based on time-consuming and laborious flow sorting using a multitude of immunophenotypic surface markers, which are potentially affected by enzymatic digestion of the source tissues. To allow for an easier, faster, and more efficient isolation of functional cross-presenting DCs from various tissues, including tumors, we used specific MACS® Tissue Dissociation Kits in combination with the gentleMACS™ Octo Dissociator with Heaters. These are optimized tools for gentle enzymatic and mechanical tissue dissociation resulting in single-cell suspensions with high viability rates and preserved cell surface marker epitopes. Additionally, we developed the CD11c MicroBeads UltraPure and the Anti-XCR1 MicroBead Kit based on MACS Technology, for subsequent enrichment of cDC subsets and specific isolation of pure XCR1+ cross-presenting DCs, respectively. Furthermore, we designed specific panels of recombinant REAfinity™ Antibodies and corresponding gating strategies to facilitate a clear phenotype analysis of DC subsets from several mouse tissues by flow cytometry. These methods will facilitate studies of the complex DC biology, which ultimately could help to develop new therapeutic strategies employing DCs for anti-tumor immune responses.





EMDS2018

25

P2THE ROLE OF COAGULATION FACTORS EXPRESSED ON MONOCYTE DERIVED MACROPHAGES

Hannes Datler(1) - Omar Sharif(1) - Andrea Vogel(1) - Martina Kerndl(1) - Julia Brunner(1) - Melanie Hofmann(1) - Laszlo Musiejovsky(1) - Christina Baumgartinger(1) - Jose Basilio(1) - Georg Obermayer(2) - Christoph J. Binder(2) - Gernot Schabbauer(1)

Institute for Vascular Biology and Thrombosis Research, Medical University of Vienna, Centre of Physiology and Pharmacology, Vienna, Austria(1) - Dept. of Laboratory Medicine, Medical University of Vienna & Center For Molecular Medicine of The Austrian Academy of Sciences, Vienna, Austria(2)

Inflammation and coagulation are intertwined physiologic processes. Monocytes and macrophages play a critical role in this interplay as they are key effectors of innate immunity that also express tissue factor (TF), the principle inducer of the extrinsic coagulation cascade as well as secrete coagulation factor VII (FVII) and X (FX). Recent seminal work has demonstrated extensive differences between tissue resident and monocyte derived macrophages (MDMs). Despite extensive literature indicating that myeloid TF is linked to thrombotic events during various diseases , like sepsis, the precise sub-population(s) of TF expressing monocyte-derived-macrophages (MDMs) and their ability herein to secrete FVII and FX remains undefined and is likely to be tissue and disease specific. Indeed, using a sterile inflammation model we could identify differences in the expression of coagulation factors between subpopulations of MDMs and resident macrophages. Notably, granulocyte-macrophage colony-stimulating factor (GM-CSF), a well-studied driver of monocyte differentiation and proliferation, heightened TF expression on specific subpopulations of infiltrating MDMs. Further, GM-CSF treated cells demonstrated augmented thrombin generation versus controls suggesting monocytes are capable of influencing the coagulation cascade. These data provide novel insight into how MDMs influence inflammation by modulating coagulation and fibrin generation on site.





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P3EVALUATION OF GM3-CONTAINING LIPOSOMES FOR ANTIGEN TARGETING TO SPLENIC CD169+ MACROPHAGES TO INDUCE ANTI-CANCER IMMUNITY

Joanna Grabowska(1) - Dieke Van Dinther(1) - Katarzyna Olesek(1) - Leoni Hoogterp(1) - Martino Ambrosini(1) - Paul R. Crocker(2) - Gert Storm(3) - Yvette Van Kooyk(1) - Joke M.M. Den Haan(1)

Amsterdam Umc, Molecular Cell Biology and Immunology, Amsterdam(1) - University of Dundee, Division of Cell Signaling and Immunology, Dundee(2) - University of Utrecht, Department of Pharmaceutics, Utrecht(3)

Liposomes are an attractive antigen delivery system and have been successfully used for vaccination strategies. We previously have shown that antibody-mediated antigen (Ag) targeting to CD169+ macrophages stimulates superior Ag-specific CD8 T cell responses. CD169, also known as sialoadhesin or siglec-1, is a sialic-acid binding lectin which has been described to bind to ganglioside GM3. Here we tested GM3-containing liposomes for their binding and uptake by murine CD169+ macrophages and their capacity to induce immune responses. As expected, CD169+ macrophages strongly and specifically bound GM3 liposomes, while macrophages from sialoadhesin knock-in mice bearing a mutation in the CD169 ligand binding pocket were incapable of binding GM3 liposomes. After intravenous administration, GM3/ovalbumin-containing liposomes specifically bound to CD169+ macrophages in a sialic acid dependent manner and stimulated ovalbumin-specific CD8 and CD4 T cell and B cell responses. Surprisingly, liposomes without GM3 also stimulated immune responses, while not binding to CD169+ macrophages. In our current studies we will also evaluate liposomes that contain the ganglioside GM1, that does not bind CD169, for their capacity to evoke an immune response. In future studies we will determine the efficacy of GM3 liposomes to target tumor antigens to CD169+ macrophages in order to induce anti-cancer immune responses.





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P4AMPLIFIED HOST DEFENSE BY TOLL-LIKE RECEPTOR-MEDIATED DOWNREGULATION OF THE GLUCOCORTICOID-INDUCED LEUCINE ZIPPER (GILZ) IN MACROPHAGES

Jessica Hoppstädter(1) - Britta Diesel(1) - Nina Hachenthal(1) - Marie Minet(1) - Petra Leidinger(2) - Christina Backes(3) - Friedrich Grässer(4) - Eckart Meese(2) - Stefano Bruscoli(5) - Carlo Riccardi(5) - Hanno Huwer(6) - Alexandra K. Kiemer(1)

Pharmaceutical Biology, Department of Pharmacy, Saarland University, Saarbrücken, Germany(1) - Human Genetics, Department of Medicine, Saarland University, Homburg, Germany(2) - Chair For Clinical Bioinformatics, Saarland University, Saarbrücken, Germany(3) - Virology, Department of Medicine, Saarland University, Homburg, Germany(4) - Pharmacology, Department of Medicine, Perugia University, Perugia, Italy(5) - Cardiothoracic Surgery, Völklingen Heart Centre, Völklingen, Germany(6)

Activation of toll-like receptors (TLRs) plays a pivotal role in the host defense of bacteria and results in the activation of NF-kappaB, leading to transcription of proinflammatory mediators. Glucocorticoid-Induced Leucine Zipper (GILZ) is an anti-inflammatory mediator, which interferes with NF-kappaB-mediated gene transcription in macrophages. Thus, we aimed to investigate the regulation and role of GILZ expression in primary human and murine macrophages upon TLR activation.Treatment with TLR agonists, e.g., Pam3CSK4 (TLR1/2) or LPS (TLR4) rapidly decreased GILZ mRNA and protein levels. GILZ downregulation required NF-kappaB-mediated upregulation of the mRNA-binding protein tristetraprolin (TTP, ZFP36), as shown by inhibitor and knockdown experiments. In return, GILZ downregulation led to an enhanced induction of proinflammatory mediators, increased phagocytic activity, and a higher capacity to kill intracellular bacteria, as shown in GILZ knockout macrophages.The TLR3 ligand polyinosinic:polycytidylic acid (Poly(I:C)) displayed no ability to decrease GILZ mRNA levels. However, GILZ protein expression was reduced by Poly(I:C)-treatment, which was paralleled by sensitization towards TLR1/2- and TLR4-agonists. A bioinformatics approach implicated more than 250 microRNAs as potential GILZ regulators. Microarray analysis revealed that the expression of several potentially GILZ-targeting microRNAs was increased after Poly(I:C) treatment in primary human macrophages. We tested the ability of 11 microRNAs to target GILZ by luciferase reporter gene assays. Within this small set, four microRNAs were confirmed as GILZ regulators, suggesting that GILZ downregulation upon TLR3 activation is a consequence of the synergistic actions of multiple microRNAs.In summary, our data indicate a dual regulation of GILZ upon TLR stimulation, which contributes to macrophage activation.





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P5THE ROLE OF DENDRITIC CELL SUBSETS IN T REGULATORY CELL INDUCTION DURING ALLERGIC AIRWAY INFLAMMATION

Emma Houlder(1) - Sheila Brown(1) - Amanda Ridley(1) - Freya Svedberg(2) - Cecilia Forss(3) - Mark Wilson(4) - Antoon Van Oosterhout(5) - Matthew Edwards(5) - Peter Cook(1) - Andrew Macdonald(1)

Mccir, University of Manchester, Manchester, United Kingdom(1) - Mccir, University of Manchester, University of Manchester, United Kingdom(2) - Mccir, Manchester, Manchester, United Kingdom(3) - Genentech Inc, Immunology Discovery, San Francisco, United Kingdom(4) - Glaxosmithkline, Respiratory R&d, Stevenage, United Kingdom(5)

Dendritic cells (DCs) have a critical role in the regulation of the immune response, in part by their induction of T regulatory cells (Tregs). Treg induction by DCs has been well characterised under tolerogenic conditions, with particular subsets of DCs able to induce Tregs by a variety of mechanisms. In contrast, much less is known about the ability of DCs to induce Tregs during ongoing inflammation. Here, we have utilised a murine model of fungal allergic airway inflammation induced by intranasal exposure to live low-dose Aspergillus fumigatus conidia to directly address these questions. Using this model, we have observed expansion of lung Foxp3+ Tregs following the development of a mixed type-2/type-17 response. Using transgenic depletion models we have shown that these Tregs are vital for control of excessive type-2 inflammation in this system. Further, we have unpicked the role of particular DC subsets in both the induction of the inflammatory (Th2/17) and regulatory (Treg) response. We have observed no contribution of conventional DC1s (cDC1s), monocyte derived DCs (moDCs) or plasmacytoid DCs (pDCs) to the inflammatory or regulatory response. Contrastingly, when MGL2+ cDC2s were depleted, a reduction in the inflammatory response was observed, with a corresponding decrease in the frequency and number of lung Foxp3+ Tregs. Ongoing work is focusing on understanding the dual role of these MGL2+ cDC2s, as well as the tolerogenic mechanisms they employ. Improved understanding of the mechanisms by which DCs can initiate Treg responses during inflammation will inform future development of innovative therapeutics for chronic type-2 disease.





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P6PTEN IN APCS REGULATES AUTOIMMUNITY BY T-CELL CHECK POINT INHIBITION

Martina Kerndl(1) - Andrea Vogel(1) - Melanie Hofmann(1) - Julia Stefanie Brunner(1) - Hannes Datler(1) - Omar Sharif(1) - Gernot Schabbauer(1)

Vascular Biology, Medical University Vienna, Vienna, Austria(1)

Exquisite mechanism(s) have evolved to protect organisms from autoimmunity. Herein, so-called checkpoint inhibitory molecules such as T-cell expressed PD-1 and its ligands, PD-L1 and PD-L2 are essential for limiting peripheral T-cell effector functions and promoting self-tolerance. Indeed, blockage of this axis during experimental autoimmune encephalomyelitis (EAE) exacerbates disease. The Phosphatidylinositol 3-kinase (PI3K) pathway is a central to key cellular processes including cell proliferation and its activity is negatively regulated by the tumor suppressor, Phosphatase and tensin homolog, (PTEN). Thus, PI3K inhibitors have been discussed as therapeutics for several cancers. Recent data indicate that this pathway plays a critical role during autoimmunity through effects on macrophage and T cell polarization, yet its impact on checkpoint inhibition remains poorly understood. To determine a potential role of PI3K herein, we utilized antigen presenting cells (APCs) from mice overexpressing or lacking myeloid specific PTEN expression (PTENTg/+ and PTENfl/fl LysMCre/+ respectively). While PTENTg/+

APCs exhibited lower basal PDL-1/2 expression versus controls, PTEN-/-APCs demonstrated augmented expression. During EAE, PTEN deficiency positively impacted regulatory T cell levels, in a manner that was intimately related to APC activity. These data indicate that PI3K activity plays a crucial role in checkpoint inhibition and suggest that studies that employ PI3K inhibitors for cancer therapeutics should examine their effects on T cell functio model.





EMDS2018

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P7THE E3 UBIQUITIN LIGASE PELLINO2 MEDIATES TLR9-INDUCED INFLAMMATION

Ewa Oleszycka(1) - Ronan Bergin(1) - Paul N. Moynagh(1,2)

Department of Biology, Maynooth University, Maynooth, Ireland(1) - Centre for Infection and Immunity, Queen’s University Belfast, Belfast, United Kingdom(2)

Ubiquitination regulates immune responses and multiple E3 ubiquitin ligases have been studied in the context of their role in myeloid cell functions in immunity. However, there are still remaining questions about the function of the E3 ubiquitin ligase Pellino2 in immune system. Therefore, the role of Pellino2 in dendritic cells (DC) and macrophages was investigated. Pellino2-deficient (Pellino2-/-) DCs stimulated with various pathogen-associated molecular patterns expressed similar levels of activation markers and secreted comparable levels of cytokines when compared to control wild-type cells. However, deletion of Pellino2 inhibited the production of pro-inflammatory cytokines by DCs following TLR9 activation. Importantly, this effect was DC-specific, as this phenotype was not observed in Pellino2-deficient macrophages. Furthermore, inflammatory response to injection of CpG, a known TLR9 agonist, was impaired in Pellino2-deficient mice. There was overall reduced cell infiltration into spleen. While T cells and B cells were recruited at similar level in both WT and Pellino2-deficient mice, the number of B220-CD3- cells was reduced in Pellino2-/- mice. When different myeloid cell populations were analysed, the effect of Pellino2 was the most profound on CD11b-F4/80+ macrophage population. Overall, this study shows that Pellino2 regulates TLR signalling in dendritic cells and also recruitment of myeloid cells and suggests it has an important role in inducing immunity. Our findings highlight a novel mechanism in regulating immune responses and increase our understanding of Pellino2 protein functions.





EMDS2018

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P8HUMAN PERIPHERAL BLOOD MONOCYTES EXCEED MONOCYTE-DERIVED MACROPHAGES AS PROSTAGLANDIN SOURCES

Sonja Rittchen(1) - Katharina Jandl(2) - Rufina Schuligoi(1) - Akos Heinemann(1)

Otto Loewi Research Center, Division of Pharmacology, Medical University of Graz, Graz(1) - Lung Vascular Research, Ludwig Boltzmann Institute, Graz(2)

During inflammation, circulating monocytes may infiltrate affected tissues, release mediators modulating inflammation or differentiate into macrophages, which likewise play a crucial role in promoting, regulating and resolving inflammatory processes. Prostaglandin D2 is an early-phase lipid mediator tightly associated with influencing inflammatory reactions. Here, we wanted to i) quantify hematopoietic PGD synthase (hPGDS) expression, the rate-limiting enzyme of PGD2 production, in human monocytes and macrophages, ii) elucidate their time-dependent involvement in elevated PGD2 levels and iii) determine the ratio of PGE2 to PGD2 release after activation with LPS/INF-γ. Human peripheral blood monocytes were obtained from healthy donors and were either used directly or differentiated into monocyte-derived macrophages in vitro. Hematopoietic PGD synthase, lipocalin-type PGD synthase, mPGES-1 and COX-2 expression was assessed on protein and mRNA level in resting and INF-γ/LPS-activated mononuclear phagocytes. PGD2 and PGE2 levels in conditioned medium were quantified by ELISA and RIA, respectively. Furthermore, murine alveolar macrophages from ovalbumin-induced allergic airway inflammation, LPS-induced acute lung injury as well as naïve mice were isolated via bronchoalveolar lavage and prostanoid production was compared. So far, we have found convincing evidence on protein and mRNA level that hPGDS is present in human monocyte-derived macrophages as well as circulating monocytes. In both cell types, INF-γ/LPS-activation did not upregulate hPGDS mRNA expression but rather downregulated its expression 24 hours after activation. Interestingly, we could detect more hPGDS expression on protein level 8 hours after activation in monocyte-derived macrophages, which indicates another regulatory step in PGD2 production. In vitro, INF-γ/LPS-activation triggered a robust PGD2 and PGE2 release, which was more pronounced in monocytes in comparison to macrophages. This study sheds more light on the role of hPGDS in human peripheral blood monocytes and macrophages and, for the first time, marks inflammatory monocytes as potent PGD2 sources.Acknowledgements: Austrian Science Fund, FWF (W1241)





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P9RAPAMYCIN INHIBITS THE EXPRESSION OF PROINSULIN CELLS IN BONE MARROW DERIVED DENDRITIC CELLS FROM DIABETIC PATIENTS. AN IN VITRO STUDY

Luisa Sambado(1) - Piero Maria Stefani(2) - Laura Valori(3) - Michele Gottardi(2) - Lucia Zanatta(4) - Matilde Cacciatore(4) - Angelo Paolo Dei Tos(4) - Agostino Paccagnella(1) - Maria Sambataro(1)

Ospedale Santa Maria Cà Foncello, Malattie Endocrine, del Ricambio e della Nutrizione, Treviso, Italy(1) - Ospedale Santa Maria Cà Foncello, Ematologia, Treviso, Italy(2) - Ospedale Santa Maria Cà Foncello, Anatomia Patologica, Treviso, Italy(3) - Ospedale Santa Maria Cà Foncello, Anatomia Patologica, Treviso, Italy(4)

IntroductionDiabetic foot is a heavy complication caused by poor circulation, neuropathic insensitivity and infected wounded feet in diabetes. Dendritic cells (DC) are sentinels in peripheral tissues, where they come into direct contact with invading pathogens and are metabolically active. In neuropathic diabetic subjects, but not in healthy controls, DC express Proinsulin (PI) marker that maybe could have a role in diabetic foot lesions.

AimWe evaluated Proinsulin expression in bone marrow-derived diabetic DC differentiated from mononuclear cells(BMMC) and their response to different stimuli in presence of glucose.

MethodsBone marrow from diabetic patients with foot lesions(n=9) were processed to obtain BMMC, seeded and cultured for 8 days in RPMI with 10% FBS,GM-CSF 50ng/mL,IL-4 50ng/mL,1%glutamine and 1% antibiotics. On day 7 cells were treated for 24h with LPS 0,1mg/mL,Glucose 30mM and/or Rapamycin 50mM. After the treatment cells were counted and analyzed by flow cytometry studying viability and the expression of Proinsulin, CD14 and dendritic markers. RESULTS:Cells number reduces on day 7 only in hyperglycemic conditions and rapamycin doesn’t reverse this reduction(138±23;87±12;85±11 respectively p<0.01). In standard medium we found: a significant decrease of CD14+cells and PI+expression (7.1±4.9vs0.7±0.5p<0.001;0.24±0.13vs0.03±0.03); a significant increase of DC+cells and DC+PI+ cells (5.5±43.5vs28.7±13 p<0.0001;0.04±0.07vs0.19±0.24 p<0.05). In glucose treated cells from T0 to T8, the behavior was reversed and rapamycin significantly inhibited the expression of PI+cells only in glucose treatment(1.5±1.2vs0.3±0.28 p<0.05)

ConclusionWe hypothize that PI expression protect from hyperglicemic-induced cell apoptosis by avoiding intracellular oxidative stress. The differentiation of PI+DC+ suggests CD14+PI+ origin. Like in vivo data, PI+increased in presence of glucose excess. In vitro probably Rapamycin changes oxidative metabolic enzymatic pathways and immunological cell replication, and this action impact on PI+cell expression perhaps in diabetic patients. Further studies are necessary to establish the real biological significance of proinsulin action in innate immune system.





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P10ROLE OF COMPLEMENT INHIBITOR FACTOR H IN MONOCYTE PHAGOCYTOSIS

Karolina Izabela Smolag(1) - Anna Maria Blom(1) - Myriam Martin(1)

Lund University, Translational Medicine, Malmo, Sweden(1)

The complement system plays a crucial role in the innate defense against pathogens, modulates adaptive immune responses and facilitates clearance of dying cells. Both excessive as well as decreased complement activa tion contribute significantly to the pathology of many diseases and recurrent infections. Considering this destructive potential, it is necessary that the system is tightly regulated by several inhibitors including factor H (FH). This protein is mainly produced in the liver, but also locally by myeloid and non-myeloid cells and circulates in blood. FH modulates various cellular functions such as neutrophil phagocytosis and adhesion of cells to bacterial surfaces. In the inflamed retina it promotes persistence of phagocytes.The aim of this study is to investigate the influence of FH on monocyte phagocytosis; firstly when being bound to the target, secondly when used as stimulus for the monocytes. FH bound to nucleosomes and apoptotic cells facilitates their phagocytosis by non-stimulated primary human monocytes. Stimulation of the monocytes with FH also changes their capacity to phagocytize nucleosomes and apoptotic cells. Phagocytosis of FH-bound targets as well as direct stimulation of monocytes with FH induces changes in the released cytokine profile. Sialic acid-binding immunoglobulin-type lectins (Siglecs) are an important family of immunoreceptors with many of the members having immunoreceptor tyrosine inhibitory motifs. We revealed that FH bound to nucleosomes induces tyrosine phosphorylation of Siglec-3 and Siglec-9 in monocytes. FH can directly bind to Siglec-9 and this interaction is independent of the presence of nucleosomes. However, FH can also bind to sialic acids that in turn interact with Siglec-9. This unconventional role of FH in removal of potential autoantigens might have a role in autoimmune diseases where problems with efferocytosis play a crucial role. The importance of these findings is also supported by strong associations between genetic alterations in FH and pathology of these diseases.





EMDS2018

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P11CELLULAR AND VIRAL FLIP PROGRAMS BROAD IMMUNE REGULATORY TRANSCRIPTION IN MONOCYTES

Alessandra Fiore(1) - Stefano Ugel(1) - Francesco De Sanctis(1) - Sara Sandri(1) - Giulio Fracasso(1) - Rosalinda Trovato(1) - Silvia Sartoris(1) - Samantha Solito(2) - Susanna Mandruzzato(2) - Fulvia Vascotto(3) - Keli Hippen(4) - Giada Mondanelli(5) - Ursula Grohmann(5) - Geny Piro(6) - Carmine Carbone(7) - Davide Melisi(7) - Rita Lawlor(8) - Aldo Scarpa(9) - Alessia Lamolinara(10) - Manuela Iezzi(10) - Matteo Fassan(11) - Silvio Bicciato(12) - Bruce Blazar(13) - Ugur Sahin(14) - Peter Murray(15) - Vincenzo Bronte(1)

University of Verona, Immunology Section, Department of Medicine, Verona, Italy(1) - University of Padova, Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, Padova, Italy(2) - University Medical Center of Johannes Gutenberg University, Tron-translational Oncology, Mainz, Germany(3) - University of Minnesota, Department of Pediatrics, Division of Blood and Marrow Transplantation, Minneapolis, United States(4) - University of Perugia, Department of Experimental Medicine, Perugia, Italy(5) - University of Verona, Department of Medicine, Digestive Molecular Clinical Oncology Research Unit, Verona, Italy(6) - University of Verona, Department of Medicine, Digestive Molecular Clinical Oncology Research Unit, Verona, Verona, Italy(7) - Arc-net Centre For Applied Research On Cancer, University and Hospital Trust of Verona, Verona, Verona, Italy(8) - University of Verona, Department of Pathology and Diagnostics, Verona, Italy(9) - University G. D’annunzio of Chieti-Pescara, Department of Medicine and Aging Science, Center of Excellence On Aging and Translational Medicine, Chieti, Italy(10) - University of Padova, Department of Medicine-dimed, Padova, Italy(11) - University of Modena and Reggio Emilia, Department of Life Sciences, Center For Genome Research, Modena, Italy(12) - University of Minnesota, of Pediatrics, Division of Blood and Marrow Transplantation, Minneapolis, United States(13) - University Medical Center of Johannes Gutenberg University, Tron-translational Oncology, Mainz, Germany(14) - Max Planck Institute of Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany(15)

Cellular and viral forms of FLIP are suppressors of death pathways that originate from TNF and related receptors. During myeloid cell development, loss of c-FLIP causes selective ablation of monocytes but not neutrophils. Using bone marrow culture systems and in vivo administration, we found that low doses of chemically-diverse chemotherapy agents selectively depleted monocytes but not neutrophils. We traced this effect to selective c-FLIP down-regulation and concomitant activation of the extrinsic apoptosis, which was rescued by ectopic expression of c-FLIP or viral FLIP from Kaposi’s sarcoma virus. However, both c-FLIP and v-FLIP induced a coincident immunosuppressive phenotype in monocytes characterized by constitutive expression of hundreds of immunomodulatory genes. When transferred into either GvHD- or tumor-bearing hosts, FLIP-expressing monocytic cells blocked systemic inflammation and inhibited adaptive immunity; moreover, circulating c-FLIP+ monocytes were associated with poor prognosis in pancreatic cancer. Therefore, FLIP modulates monocytic cells in multiple ways: by controlling cell survival and by broadly altering immunoregulatory gene expression to favor immunosuppression.





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P12DISCOVERING NOVEL IMMUNE-MODULATORY GLYCANS USING HIGH-THROUGHPUT SCREENING STRATEGIES

Meshal Alobaid(1) - Sarah-Lane Richards(2) - Mathew Gibbson(3) - Morgan Alexander(4) - Amir Ghaemmaghami(5)

University of Nottingham, Phd Student Immunology, Nottingham(1) - Warwick University, Phd Department of Chemistry, Warwick(2) - Warwick University, Professor of Chemistry Department of Chemistry and Warwick Medical School, Warwick(3) - University of Nottingham, Professor of Biomedical Surfaces at The School of Pharmacy, Nottingham(4) - University of Nottingham, Professor Immunology & Immuno-bioengineering, Faculty of Medicine, Nottingham(5)

IntroductionIn this project we apply high throughput screening strategies to investigate immune modulatory properties of a combinatorial library of synthetic glycans with a particular focus on the ability of different glycans in modulating key functional properties of human Dendritic cells (DCs). We hypothesise that glycans with distinct structures are able to promote generation of immune stimulatory or regulatory DCs with the ability to initiate or suppress immune responses. Here in we look at the immunomodulatory effects of synthetic libraries consisting of combinations of mannose and galactose in two isoforms in a plate bound format on LPS stimulated DC. DCs immunogenicity is assessed using Indoleamine 2,3-dioxygenase (IDO) enzymatic activity and cytokine profile as primary readout.

ResultsPlate bound glycans are able to significantly reduce IDO activity where both isoforms of the same glycans is combined however less reductions can be seen in combinations between mannose and galactose isoforms. Furthermore, a selection of glycans also suppressed IL-12 and significantly increased IL10 production compared to LPS alone.

ConclusionsCollectively these observations show the potential immunomodulatory effects of glycans in priming DCs and skewing immune responses towards different functional phenotypes. Future work will focus on looking at downstream effects on DC-T cell co cultures. Optimised combination of different glycans could provide a powerful tool for immune modulation with potential applications in vaccination, cancer and a host of inflammatory diseases.

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P13MACROPHAGE TARGETING BY THE CYTOTOXIC NATURAL PRODUCT THIOHOLGAMIDE A AS AN ANTICANCER PRINCIPLE

Charlotte Dahlem(1) - Maria Lopatniuk(2) - Jessica Hoppstädter(1) - Sonja M. Kessler(1) - Britta Diesel(1) - Andriy Luzhetskyy(2) - Alexandra K. Kiemer(1)

Pharmaceutical Biology, Saarland University, Saarbruecken, Germany(1) - Pharmaceutical Biotechnology, Saarland University, Saarbruecken, Germany(2)

Tumor-associated macrophages (TAMs) are a key component of the tumor microenvironment and can support tumor growth and metastasis. One crucial driver of TAM polarization are apoptotic cells, which initiate a functional program in TAMs that promotes angiogenesis and cell proliferation. As classical chemotherapeutics induce apoptosis, improved therapies should not only focus on malignant cells, but also target their interaction with the microenvironment. In fact, the natural products trabectedin and doxorubicin have shown to act by targeting not only tumor cells but also macrophages. Thus, we investigated the effects of the natural compound thioholgamide A and its derivates on both cell types.All tested compounds showed distinct cytotoxic effects in different cancer cell lines (A549, HuH7, HCT116). We also observed antiproliferative effects in A549 and HuH7 cells at sub- toxic concentrations as shown by impedance measurement. In macrophages thioholgamide treatment at concentrations similar to those active against tumor cells resulted in cell death. We then determined differences of the cytotoxic potential of thioholgamide on differently polarized macrophages, i.e., human monocyte-derived macrophages polarized in vitro towards an M1, M2 or TAM-like phenotype. Thioholgamide was more effective in killing M2 and TAM-like than M1 macrophages. To investigate potential thioholgamide-induced effects on macrophage polarization, we determined alterations in gene expression of treated cells. Thioholgamide shifted gene expression of M2 cells towards a pro-inflammatory M1 state, as shown by elevated expression of TNF-α, IL-6, and IL-12, while the anti-inflammatory cytokine IL-10 was suppressed. Ongoing investigations include effects of the compounds on cell-cell interactions between monocytes and tumor cells in a 3D spheroid model.Taken together, besides its action on cancer cells thioholgamide A affects tumor-associated macrophages and might, therefore, be a promising new therapeutic agent.

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P14HYPERTHERMIC TREATMENT AT 56°C INDUCES TUMOR-SPECIFIC IMMUNE PROTECTION IN A MOUSE MODEL OF PROSTATE CANCER IN BOTH PROPHYLACTIC AND THERAPEUTIC IMMUNIZATION REGIMENS

Francesco De Sanctis(1) - Sara Sandri(1) - Matteo Martini(1) - Marta Mazzocco(1) - Alessandra Fiore(1) - Rosalinda Trovato(1) - Stefano Garetto(2) - Davide Brusa(3) - Stefano Ugel(1) - Silvia Sartoris(1)

University of Verona, Immunology Section, Department of Medicine, Verona, Italy(1) - University of Torino, Department of Internal Medicine, Torino, Italy(2) - University of Torino, Department of Internal Medicine, Torino, Italy(3)

Most active cancer immunotherapies able to induce a long-lasting protection against tumours are based on the activation of tumour-specific cytotoxic T lymphocytes (CTLs). Cell death by hyperthermia induces apoptosis followed by secondary necrosis, with the production of factors named “danger associated molecular pattern” (DAMP) molecules (DAMPs), that activate dendritic cells (DCs) to perform antigen uptake, processing and presentation, followed by CTLs cross priming. In many published studies, hyperthermia treatment of tumour cells is performed at 42-45°C; these temperatures mainly promote cell surface expression of DAMPs. Treatment at 56°C of tumour cells was shown to induce DAMPs secretion rather than their cell surface expression, improving DC activation and CTL cross priming in vitro. Thus we tested the relevance of this finding in vivo on the generation of a tumour-specific memory immune response, in the TRAMP-C2 mouse prostate carcinoma transplantable model. TRAMP-C2 tumour cells treated at 56°C were able not only to activate DCs in vitro but also to trigger a tumour-specific CTL-dependent immune response in vivo. Prophylactic vaccination with 56°C-treated TRAMP-C2 tumour cells alone provided protection against TRAMP-C2 tumour growth in vivo, whilst in the therapeutic regimen, control of tumour growth was achieved combining immunization with adjuvant chemotherapy. .

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P15REPROGRAMMING OF TUMOR-ASSOCIATED MACROPHAGES THROUGH ID4-DEPENDENT PARACRINE ACTIVITY OF BREAST CANCER CELLS

Sara Donzelli(1) - Elisa Milano(1) - Magdalena Pruszko(2) - Andrea Sacconi(1) - Silvia Masciarelli(3) - Enzo Gallo(4) - Alicja Zylicz(2) - Francesco Fazi(3) - Giovanni Blandino(1) - Giulia Fontemaggi(1)

Irccs Regina Elena National Cancer Institute, Oncogenomic and Epigenetic Unit, Rome, Italy(1) - International Institute of Molecular and Cell Biology In Warsaw, Department of Molecular Biology, Warsaw, Poland(2) - Sapienza University of Rome, Section of Histology & Medical Embryology, Rome, Italy (3) - Irccs Regina Elena National Cancer Institute, Pathology Dept, Rome, Italy(4)

Breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (Inhibitors of Differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like and triple-negative breast cancer. Moreover, ID4 favors angiogenesis by enhancing the expression of pro-angiogenic cytokines IL-8, CXCL1, and VEGF in breast cancer cells. Here, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer.We determined that ID4 and macrophage marker CD68 protein expression are significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 mRNA levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signaling, the activation of an angiogenic program in macrophages. This program includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine Granulin (GRN), whose enhanced expression in macrophages confers increased angiogenic potential. of note, induction of ID4 mRNA and protein is also observed in macrophages cultured with conditioned medium from breast cancer cells expressing high ID4 levels. On the contrary, ID4 induction is not observed upon treatment with other macrophage-stimulating factors, such as LPS and IL-4/IL-13. Functionally, induction of ID4 in macrophages reduces the expression of macrophage differentiation markers.

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P16ADENOSINE REPOLARISES HUMAN MACROPHAGES TO AN IMMUNOSUPPRESSIVE PHENOTYPE VIA BOTH A2A AND A2B ADENOSINE RECEPTORS

A.M. Jackson(1) - A.P. Kaur(1) - T. Musarrat(1) - P.M. Patel(1) - S.J. Hill(2) - H.A. Franks(1)

University of Nottingham, Academic Clinical and Translational Oncology, Nottingham, United Kingdom(1) - University of Nottingham, School of Life Sciences, Nottingham, United Kingdom(2)

Macrophages are a key component of solid tumours and their polarisation markedly impacts on tumour microenvironment immunogenicity. Therefore, targeting macrophages to restore immunogenicity is a viable approach to improve immunotherapy. High levels of adenosine in tumours are immunosuppressive, and whilst the adenosine pathway is a novel immunotherapeutic target its impact on human macrophages remains to be fully understood. We therefore studied the effect of adenosine on macrophage activation and polarisation and addressed the role of the A2A and A2B adenosine receptors.Differentiation of macrophages from CD14+ monocytes was affected by the adenosine analogue NECA, producing less IL-12 (GMCSF-differentiated macrophages (GMCSF-mac) and more IL-10 (GMCSF-mac and MCSF-differentiated macrophages (MCSF-mac)). Mean IL-12:IL-10 ratio reduced from 3.1:1 in control GMCSF-mac to 0.90:1 in NECA-conditioned GMCSF-mac (p<0.05). Mean fold change in IL-10 for NECA-conditioned MCSF-mac was x1.9 compared to control MCSF-mac (p<0.05). Consequently, NECA-conditioned MCSF-mac differentially signalled to PHA-stimulated lymphocytes which produced lower levels of IFNγ (mean 46% of production by lymphocytes co-cultured with control MCSF-mac, p<0.05) without altering their proliferation. Importantly, the alteration in IL-12:IL-10 production and suppression of IFNγ production by co-cultured lymphocytes was reversed by blockade of both A2A and A2B receptors during differentiation (SCH442416 and PSB603 respectively).The adenosine pathway not only impaired macrophage differentiation but also the function of otherwise healthy macrophages. Exposure of control GMCSF-mac to increasing concentrations of NECA immediately prior to activation suppressed IL-12 and increased IL-10 production (both p<0.00001). This effect was mediated by A2A and A2B as simultaneous blockade of both receptors was required to prevent the effect of NECA.Thus, we have shown that adenosine polarises human macrophages towards an immunosuppressive phenotype. This leads to a suppression of IFNγ responses, a key cytokine for anti-tumour immune responses. This effect is mediated via both the A2A and A2B adenosine receptors, suggesting that blockade of both receptors may be required to repolarise macrophages within the tumour microenvironment. This is in contrast to lymphocytes, where the effect of adenosine is mediated via the A2A adenosine receptor only.

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P17MYELOID MTORC1 AND ITS INVOLVEMENT IN COLITIS ANDCOLORECTAL CANCER

Stephanie Deborah Fritsch(1) - Birgit Schütz(1) - Nyamdelger Sukhbaatar(1) - Michaela Lang(2) - Christoph Magnes(3) - Jakob Weiszmann(4) - Markus Hengstschläger(1) - Thomas Weichhart(1)

Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria(1) - Department of Internal Medicine Iii, Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria(2) - Joanneum Research, Forschungsgesellschaft Mbh, Graz, Austria(3) - Department of Ecogenomics and Systems Biology / Vienna Metabolomics Center (vime), University of Vienna, Vienna, Austria(4)

The gut is responsible for digestion, absorption and metabolism of dietary nutrients. It has the highest cell turnover of any tissue, which is supported by polyamines, aliphatic compounds essential for many biological functions. A dysregulation of this highly regulated homeostasis can promote inflammatory bowel diseases (IBDs), which are the main risk factors for generating colitis-associated colorectal cancer. The mammalian target of rapamycin (mTOR) has a central role in the effector functions of immune cells and both mTOR complexes are involved in colorectal cancer (CRC) formation. Little is known about the cell-specific mTOR functions in immune cells during CRC. In our mouse model, mTORC1 is constitutively active in macrophages by deletion of the gene Tsc2 (Tsc2fl/flLyz2cre/+ mice). These macrophages tend to be of the anti-inflammatory M2 type, show increased proliferation and present increased enzymes of polyamine anabolism via an mTORC1-mediated mechanism. In fact, significantly increased polyamine levels are seen in bone marrow derived macrophages as well as in colon samples of healthy Tsc2fl/flLyz2cre/+ mice. Interestingly, these mice are largely protected from developing DSS-induced colitis and are able to maintain a normal gut structure even after the AOM/DSS-induced colitis-associated CRC model with the net result of a decreased tumor burden. Since polyamines are known to have anti-inflammatory properties and to be fundamental in gut regeneration and healing processes, we hypothesize their involvement in the resistance to the DSS-induced colitis. The aim of our study is to understand the underlying mechanisms responsible for the protection from colitis in Tsc2fl/flLyz2cre/+ mice. This might help to improve treatments for IBDs and reduce the formation of colorectal cancer, one of the commonest types of cancer in western industrialized countries.

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P18DEFINING THE IMMUNOMODULATORY EFFECTS OF IMM-101: A PROMISING, NOVEL CO-THERAPY FOR CANCER

Alicia Galdon(1) - James Crooks(1) - Sheila Brown(1) - Laura Rosa Brunet(2) - Andrew Macdonald(1)

Manchester Collaborative Centre For Inflammation Research, The University of Manchester, Manchester, United Kingdom(1) - Rb Consulting Ltd., -, London, United Kingdom(2)

IMM-101 is a whole-cell preparation of heat-killed Mycobacterium obuense (NCTC13365) currently undergoing clinical evaluation for the treatment of advanced pancreatic adenocarcinoma. With a 5-year survival rate of less than 5%, novel treatments for pancreatic cancer are a major clinical need. In a phase II trial, treatment with IMM-101 in combination with gemcitabine (one of the current standard-of-care options for pancreatic cancer) increased median survival from 4.4 months to 7 months in patients with metastasis. Pre-clinical studies in syngeneic murine models have also found significantly decreased tumour burden when IMM-101 is administered in combination with the checkpoint inhibitors anti-PD-1, compared to anti-PD-1 alone. Establishing the mechanism by which IMM-101 causes this effect is key for advancing the use of this bacterial immunomodulator as a cancer therapy. We have found that exposure to IMM-101 significantly increases the activation status of both human and murine dendritic cells (DCs) in vitro, enhancing their ability to process and present antigen to CD4+ T cells. Further characterisation in vivo showed that IMM-101 primed DCs induced strong IFN-g production by a variety of different immune cell populations, including CD4+, CD8+, NK, NKT and gd T cells. Intriguingly, IFN-g induction occurred independently of IL-12p70 secretion by IMM-101 primed DCs. Commensal bacterial involvement in the response induced by IMM-101 has also been ruled out, with germ-free animals showing equivalent responses to SPF controls after direct IMM-101 injection, and also following transfer of IMM-101 treated DCs. These data shed some light on the potential role of IMM-101 in promoting anti-tumour immune responses through the activation of DCs and induction of anti-tumour cytokines. Ongoing work is focusing on determining the mechanisms by which IMM-101 influences the activation and function of specific DC subsets, and their induction of IFN-g, in vivo.

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P19INACTIVATION OF MTORC2 IN MACROPHAGES IS A SIGNATURE OF COLORECTAL CANCER THAT PROMOTES TUMORIGENESIS

Karl Katholnig(1) - Birgit Schütz(1) - Stephanie Deborah Fritsch(1) - David Schörghofer(1) - Monika Linke(1) - Nyamdelger Sukhbaatar(1) - Julia Maria Matschinger(1) - Daniela Unterleuthner(1) - Martin Hirtl(1) - Michaela Lang(2) - Merima Herac(3) - Andreas Spittler(4) - Andreas Bergthaler(5) - Gernot Schabbauer(6) - Helmut Dolznig(1) - Mark A. Magnuson(7) - Mario Mikula(1) - Stefanie Horer(1) - Thomas Weichhart(1)

Institute of Medical Genetics, Center of Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria(1) - Department of Internal Medicine Iii, Division of Gastroenterology and Hepatology, Vienna, Austria(2) - Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria(3) - Core Facility Flow Cytometry & Surgical Research Laboratories, Medical University of Vienna, Vienna, Austria(4) - Cemm Research Center For Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria(5) - Institute For Physiology, Center For Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria(6) - Department of Molecular Physiology and Biophysics and Center For Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, United States(7)

The mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) is a novel promising anti-cancer target due to its critical role in proliferation, apoptosis and metabolic reprogramming of cancer cells. However, the activity and function of mTORC2 in malignant tissue in vivo is insufficiently explored. Surprisingly, in primary human and mouse colorectal cancer (CRC) samples, mTORC2 signaling could not be detected in tumor cells. In contrast, only macrophages in tumor-adjacent areas showed mTORC2 activity that was downregulated in stromal macrophages inside of human and mouse tumor tissues. Functionally, inhibition of mTORC2 by specific deletion of Rictor in macrophages stimulated tumorigenesis in a colitis-associated CRC mouse model. This phenotype was driven by a pro-inflammatory metabolic reprogramming of mTORC2-deficient macrophages that promoted colitis via the cytokine SPP1/osteopontin to stimulate tumor growth. In human CRC patients, high SPP1 levels and low mTORC2 activity in tumor-associated macrophages correlated with a worse clinical prognosis. Treatment of mice with a second generation mTOR inhibitor that inhibits mTORC2 and mTORC1 exacerbated experimental colorectal tumorigenesis in vivo. In conclusion, mTORC2 activity is confined to macrophages in colorectal cancer and prevents tumorigenesis. These results suggest activation but not inhibition of mTORC2 as a therapeutic strategy for colorectal cancer therapy.

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P20CHARACTERIZATION OF A NEW IN VITRO MODEL FOR TUMOR-ASSOCIATED MACROPHAGES AND COMPARISON WITH PATIENT-DERIVED MACROPHAGES FROM LUNG TUMOR TISSUE

Jessica Hoppstädter(1) - Anna Dembek(2) - Nathalie Wirth(3) - Daniela Oster(1) - Gil Gasparoni(4) - Martin Simon(5) - Marcel H. Schulz(6) - Hanno Huwer(7) - Alexandra K. Kiemer(8)

Pharmaceutical Biology, Saarland University, Saarbrücken, Germany(1) - Pharmaceutical Biology, Saarland University, Saarbrücken, Germany(2) - Computational Biology and Applied Algorithmics, Max Planck Institute For Informatics, Saarland Informatics Campus, Saarbrücken, Germany(3) - Genetics/epigenetics, Saarland University, Saarbrücken, Germany(4) - Molecular Cell Dynamics, Saarland University, Saarbrücken, Germany(5) - Cluster of Excellence In Multimodal Computing and Interaction, Saarland University and Max Planck Institute For Informatics, Saarland Informatics Campus, Saarbrücken, Germany(6) - Cardiothoracic Surgery, Völklingen Heart Centre, Völklingen, Germany(7) - Pharmaceutical Biology, Saarland University, Saarbrücken, Germany(8)

Lung cancer represents the most common cause of cancer-related deaths worldwide. In general, tumor-associated macrophages (TAMs) promote tumor growth. Understanding the process of macrophage polarization in the tumor microenvironment might lead to new therapeutic approaches. Therefore, we aimed to characterize in vitro differentiated TAM-like macrophages and compared them to patient-derived TAMs from lung tumors. In order to mimic the tumor microenvironment, human monocyte-derived macrophages were exposed to A549 lung tumor cell supernatant. These TAM-like cells were then compared with M1 (LPS/IFN-γ)- or M2 (IL-10, IL-4)-polarized macrophages. Flow cytometric analysis showed that TAM-like macrophages express surface markers which have previously been reported to be characteristic for an M2-like phenotype. To compare this cell model with in vivo differentiated macrophages, we isolated primary TAMs and autogenetic alveolar macrophages (AMs) from patient-derived lung tissues. We compared both macrophage types from three adenocarcinoma patients at comparable age and cancer stage using paired-end mRNA-Seq. As expected, we found previously described markers of M2 polarization, such as matrix metalloproteinases and angiogenesis-related genes to be upregulated in TAMs. Interestingly, the expression of lipid metabolism-associated genes was significantly altered. Furthermore, our data suggested that TAM-like cells are a suitable model for primary in vivo differentiated TAMs, since similar genes were differentially expressed. In summary, we generated a cheap and easy to set up tool to study the tumor-associated macrophage polarization processes.

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P21NEW MONOCYTE ATTRACTING AND PRO-ANGIOGENIC FACTOR YKL-39 IS PRODUCED BY TUMOR-ASSOCIATED MACROPHAGES IN HUMAN BREAST CANCER AND IS INDICATIVE FOR INCREASED METASTASIS

Tengfei Liu(1) - Irina Larionova(2) - Vladimir Riabov(1) - Nikolai Litviakov(3) - Marina Zavyalova(3) - Andrew Flatley(4) - Elisabeth Kremmer(4) - Nadezhda Cherdyntseva(3) - Harald Klüter(1) - Julia Kzhyshkowska(1)

University of Heidelberg, Medical Faculty Mannheim, Institute of Transfusion Medicine and Immunology, Mannheim, Germany (1) - Tomsk State University, Laboratory For Translational Cellular and Molecular Biomedicine, Mannheim, Germany (2) - Tomsk State University, Laboratory For Translational Cellular and Molecular Biomedicine, Tomsk, Russian Federation (3) - Helmholtz Zentrum München, Institute of Molecular Immunology, Munich, Germany (4)

In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Using confocal microscopy we identified that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of both gene expression and production of YKL-39 in human primary macrophages. Using affinity chromatography we found, that stabilin-1 directly interacts with YKL-39 via extracellular F7 domain. Further we demonstrated that stabilin-1 acts as sorting receptor for targeting YKL-39 into the secretory pathway. Using ex vivo migration assay we found that purified YKL-39 acted as a strong chemotactic factor for primary human monocytes. In vitro tube formation assays demonstrated that YKL39 is strongly induces angiogenesis. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can reduce monocyte recruitment and angiogenesis, and as consequence, the risk of metastasis in breast cancer patients.

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EMDS2018

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P22THE CHEMOKINE CCL17 SHAPES THE TUMOR MICROENVIRONMENT IN MULTIPLE MOUSE MODELS OF CANCER

Rebecca Metzger(1) - Anne Krug(1)

Institute for Immunology, University of Munich, Munich, Germany(1)

Myeloid cells are critically involved in the pathophysiology of cancers. CCL17, a chemokine of the C-C family is known to be expressed by CD11b+ dendritic cells and to play a role in inflammatory processes, such as colitis. Using a CCL17-GFP-reporter/knock-in mouse we investigated the role of CCL17 in colon- and melanoma-cancerogenesis and find that CCL17 promotes tumor initiation and -progression. CCL17–deficient mice developed less tumors in the AOM/DSS-induced model of colitis-associated cancer and in syngeneic subcutaneous tumor models (MC38, B16) tumor growth was reduced in the absence of CCL17. Our results from the colitis-associated cancer model indicate T- and B-cell independent mechanisms, as the observed phenotype was conserved in Rag-deficient mice. In the tumor microenvironment macrophage subsets were found to express CCL17, while its expression is limited to dendritic cells in the steady state. Intriguingly, the frequencies and phenotype of tumor associated macrophage (TAM) subsets were significantly altered in the absence of CCL17. Regulatory macrophages, marked by the expression of MMR (CD206) were decreased in tumors of CCL17 deficient mice. IL-4 polarized macrophages generated from CCL17-deficient bone marrow cells in vitro showed decreased expression of Arginase-1 and increased secretion of TNFα, confirming our in vivo findings. Thus, CCL17 supports regulatory TAMs and thereby promotes an immunsuppressive tumor microenvironment.

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EMDS2018

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P23TRANSCRIPTOMICS LANDSCAPE OF NECROPTOSIS GENES IS ASSOCIATED WITH DENDRITIC CELLS INFILTRATION: A PAN-CANCER STUDY OF 5,451 PRIMARY SOLID TUMORS

Lorenzo Nicolè(1) - Tiziana Sanavia(2) - Rocco Cappellesso(3) - Ambrogio Fassina(1)

University of Padova, Department of Medicine, Padova, Italy (1) - Harvard Medical School, Department of Biomedical Informatics, Boston, United States (2) - University of Padova, Departement of Medicine, Padova, Italy (3)

AimNecroptosis (NPC) is a form of programmed cell death that culminates with the rupture of the cell membrane followed by the releasing of cellular elements. Evidence showed that tumors with high expression of NCP-related genes are associated with high cytotoxic CD8+ T-cell infiltrates, mediated by signalling from Dendritic (DC) and CD4+ T-cells. This study shows a pan-cancer view of the relationship between NCP and immune infiltration and their prognostic relevance across 24 cancer types from The Cancer Genome Atlas (TCGA).

MethodsGene expression RNA-seq data from 5,451 primary solid tumors were considered, excluding cases with treatments before surgery and with residual tumor. A deconvolution algorithm was used to estimate the level of tumor-infiltrating immune cells in each RNA-seq sample, considering the populations: B-cells, CD4 T-cells, CD8 T-cells, Macrophages and DC. For each immune population, the relative infiltration score was dichotomized at low and high infiltration using the 25th and 75h percentiles, respectively. Logistic regression and likelihood ratio test were applied to 163 genes belonging to Necroptosis pathway from KEGG database to test whether they are significantly associated to the infiltration of a specific immune population. FDR-adjusted p-values <5% were considered statistically significant. The prognostic relevance of the NCP genes significantly correlated with the infiltration was evaluated by Cox regression and log-rank test.

ResultsDC and CD4+ T-cells showed the highest number of cancer types (8) reporting more than half genes of NCP pathway significantly correlated with their infiltration. CD8+ T-cell infiltration correlated with >50% of NCP genes in 5 of these 8 cancer types: Kidney-Renal, Breast, Prostate, Pancreatic and Thyroid tumors. DC also showed the highest number of NCP genes (69) correlated with their infiltration in more than half of the analyzed cancer types, including the main genes involved in NCP execution: RIPK1, RIPK3, MLKL and CFLAR. 60 and 58 of these genes showed a prognostic relevance (p<5%) for overall and disease-free survival in at least one cancer type, respectively.

ConclusionNCP has a relevant role in eliciting immune response against tumor through DC-mediated immunity in specific cancer types.

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EMDS2018

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P24MACROPHAGES ADAPT TO THE METASTATIC NICHE IN PANCREATIC CANCER BY SENSING THE STIFFNESS

Sebastian Nielsen(1) - Edward Horton(1) - Giorgia Maltese(1) - Chang-Il Hwang(2) - James Mcconnell(3) - Michael Sherratt(3) - Janine Erler(1)

University of Copenhagen, Biotech Research and Innovation Centre, Copenhagen (1) - Cold Spring Harbor Laboratories, Cold Spring Harbor Laboratories, New York (2) - University of Manchester, University of Manchester, Manchester (3)

Pancreatic cancer has the worst prognosis of all cancer types, partly due to late detection at advanced stages of the disease, when patients already have distant metastases [1]. The presence of macrophages in the pancreatic tumour microenvironment correlates with reduced patient survival, and macrophages are recruited to the liver after the dissemination of pancreatic cancer cells (PCC), where they promote metastatic colonisation by orchestrating the establishment of a hospitable niche [2]. The extracellular matrix (ECM) is a complex network of proteins, that provides cells with a structural framework. ECM composition and rigidity are modified during tumour progression [3], however, the interplay between macrophages and changes in ECM structure at the metastatic site is not known. The aim of this project is to identify ECM-dependent metastasis-promoting functions of macrophages that can be targeted therapeutically to block metastatic progression in pancreatic cancer. Using a mouse model of liver metastasis, we demonstrate an increase in the number of macrophages in late stage metastases compared to earlier time points, and that the majority of macrophages are localised in regions with a high content of fibrillary collagen. By culturing bone marrow-derived macrophages on collagen-coated polyacrylamide gels with different stiffness (1, 8 or 25 kPa), we determine that different kinase pathways are activated in response to increased stiffness. From our kinase profiling, we identified a panel of available tyrosine kinase inhibitors and are now testing if any of these can suppress the stiffness-dependent functions of macrophages in vitro and inhibit the metastatic development in the liver using our mouse model.

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EMDS2018

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P25LEUKOCYTE-MIMETIC NANOPARTICLES TO TARGET INFLAMMATION

Anna Pasto(1) - Manuela Sushnitha(2) - Jonathan O. Martinez(3) - Roberto Molinaro(4) - Ennio Tasciotti(3)

Istituto Oncologico Veneto-IRCCS,, Department of Surgery, Oncology and Gastroenterology, Padova, Italy(1) - Rice University, Department of Bioengineering, Houston, Tx, United States(2) - Houston Methodist Research Institute, Center For Biomimetic Medicine, Houston, Tx, United States(3) - Harvard Medical School, Department of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, Ma, United States(4)

Cellular recognition is orchestrated by surface markers that mediate receptor–ligand interactions. These interactions are particularly important for circulating immune cells as they allow them to target inflamed endothelium, migrate across the vessel wall, and reach the tumor parenchyma where they activate a specific response. Inspired by leukocytes’ tropism and regulation of inflammatory response, we developed the leukosomes, leukocyte-based biomimetic NPs synthesized from purified membrane proteins extracted from circulating macrophages. In a mouse model of triple negative breast cancer, we proved the superior ability of leukosomes to: 1- escape macrophage sequestration and delay clearance from liver and spleen; 2- target inflamed endothelium and adhere to activated cells; 3- accumulate in the tumor microenvironment upon extravasation from the vessels; 4- modulate the gene expression of pro and anti-inflammatory cytokines; 5- induce the polarization of tumor associate macrophages.Treatment of breast cancer bearing mice with docetaxel-loaded leukosomes resulted in delay of tumor growth and overall increased survival, compared to drug-loaded liposome and free drug treatment. Interestingly, the administration of empty leukosomes was associated with a 15-fold increase of tumor targeting after only one hour from NP injection. We demonstrated that most of leukosomes’ emerging functions are attributed to the expression of key surface markers such as: CD45, CD47, Mac-1 and LFA-1. Indeed, leukosomes’ incubation with anti-CD45 antibody resulted in a 60% increase in liver and spleen accumulation, whereas blockage of LFA-1, resulted in a 95% decrease in tumor accumulation. In conclusion, our results demonstrated that leukosomes represent a promising delivery platform to target and treat inflammatory diseases. Leukosomes shift the original concept of inert nanocarrier to a more sophisticated entity that combines synthetic components with biologic and cell-like properties derived from the molecular machinery of the cell surface.

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EMDS2018

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P26CHARACTERIZATION OF CIRCULATING AND TUMOR-INFILTRATING MYELOID-DERIVED SUPPRESSOR CELLS IN PATIENTS AFFECTED BY PANCREATIC DUCTAL ADENOCARCINOMA

Sara Sartori(1) - Rosalinda Trovato(1) - Alessandra Fiore(2) - Francesco De Sanctis(3) - Rosalba Giugno(4) - Luciano Cascione(5) - Salvatore Paiella(6) - Francesca Hofer(1) - Ornella Poffe(1) - Cristina Anselmi(1) - Tiziana Cestari(1) - Samantha Solito(7) - Susanna Mandruzzato(8) - Boris Rusev(9) - Silvia Sartoris(1) - Rita Teresa Lawlor(10) - Aldo Scarpa(9) - Claudio Bassi(6) - Vincenzo Bronte(1) - Stefano Ugel(1)

University of Verona, Department of Medicine, Verona, Italy(1) - Max Planck Institute of Biochemistry, Immunoregulatory Department, Munich, Germany(2) - Verona, Department of Medicine, Verona, Italy(3) - University of Verona, Department of Computer Science, Verona, Italy(4) - Institute of Oncology Research, Lymphoma & Genomics Group, Bellinzona, Switzerland(5) - University of Verona, Department of Surgery and Oncology, Verona, Italy(6) - University of Verona, Centro Piattaforme Tecnologiche (CPT), Verona, Italy(7) - University of Padova, Department of Surgery, Oncology and Gastroenterology, Padova, Italy(8) - University of Verona, Department of Pathology and Diagnostics, Verona, Italy(9) - University and Hospital Trust of Verona, Arc-net Centre For Applied Research on Cancer, Verona, Italy(10)

Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with an overall 5-year survival rate of less than 5%. Newly discovered evidences clearly demonstrate that PDAC cells release high amount of pro-inflammatory metabolites that induce a marked alteration of normal hematopoiesis favoring the expansion and accumulation of myeloid-derived suppressor cells (MDSC). MDSC are an heterogeneous cell population that negatively regulates the immune response during cancer progression, inflammation and infection. The MDSC critical impact on tumor progression has been proved in a large number of cancers; in fact, the MDSC presence and frequency in blood of cancer patients generally correlate with poor clinical outcome and response to therapy. However, human MDSC are usually identified by cytofluorimetric analysis that does not depict the real biological functionality of this cell population. Here, we applied the recent approved criteria to standardize MDSC enumeration in PDAC patients demonstrating that patients with high levels of MDSC in the peripheral blood present a significant aggressive disease and poor survival. Moreover, we validated the immunosuppressive ability of human monocytes CD14+ cells isolated from PBMCs of PDAC patients by in vitro test and we performed a genetic analysis of the immunosuppressive MDSC fraction to discover new putative functional markers. Finally, we demonstrated the presence of MDSCs also within the pancreatic tumor masses, which inversely correlated with T cells frequency. Collectively, our results clearly depict the critical role of MDSC on fueling an immunosuppressive environment during PDAC progression.

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EMDS2018

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P27REGULATION OF ARGINASE1 AND INDUCIBLE NITRIC OXIDE SYNTHASE IN SALMONELLA TYPHIMURIUM INFECTED BONE MARROW DERIVED MACROPHAGES

Natascha Brigo(1) - Stefanie Dichtl(1) - Christa Pfeifhofer-Obermair(1) - Piotr Tymoszuk(1) - Markus Seifert(1) - Günter Weiss(1)

Department of Internal Medicine Ii, Medical University of Innsbruck, Innsbruck, Austria(1)

Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) are potent inducers of inducible nitric oxide (NO) synthase (iNOS) which is central for the control of Salmonella enterica serovar Typhimurium (S. Typhimurium) by macrophages. Two types of macrophages, inflammatory M1 macrophages expressing iNOS and anti-inflammatory M2 macrophages expressing Arginase1 (Arg1), are involved in the immune response against S. Typhimurium. High expression of Arg1, which cleaves L-arginine, the substrate of iNOS, impairs host control of infection with intracellular microbes. Herein, we investigated the protective effects of TNFα and IFNγ on iNOS and Arg1 expression in macrophages and how this impacts on the immune control of Salmonella infection in vitro. Bone marrow derived macrophages (BMDMs) generated from C57BL/6 WT mice upon M-CSF stimulation for six days were infected with S. Typhimurium for one hour. Cells were then stimulated with either Interleukin-4 (IL-4), IFNγ, or TNFα and combinations thereof for various time points. mRNA expression of Arg1 and iNOS was analyzed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Furthermore, bacterial load of infected BMDMs was analyzed by FACS staining and sorting as well as plating lysates on LB agar plates for determining colony forming units.We observed that IL-4 is a potent inducer of Arg1 expression in a time dependent manner, which is more pronounced in infected than in uninfected BMDMs. Accordingly, inducible Arg1 expression was significantly suppressed by IFNγ whereas TNFα had only little effect in S. Typhimurium infected BMDMs. In a comparable fashion IFNγ was the most potent inducer of iNOS expression as compared to TNF, and IL-4 blocked the latter effect more potently. Surprisingly, high iNOS expression did not translate into improved control of intracellular Salmonella proliferation and IL-4 stimulation even resulted in reduction of bacterial numbers. We are currently investigating the mechanisms underlying regulation of iNOS and Arg1 by these cytokines through transcriptional and epigenetic regulation.

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EMDS2018

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P29AN IN VITRO APPROACH TO STUDY MACROPHAGE HYPERPLASIA DURING P. FALCIPARUM MALARIA

Jill Dalimot(1) - Thomas Klei(1) - Ryanne Arisz(1) - Judy Geissler(1) - Guillaume Bouyer(2) - Stéphane Egée (2) - Robin Van Bruggen(1)

Sanquin, Blood Cell Research, Amsterdam, Netherlands(1) - Station Biologique, Compared Physiology of Erythrocytes, Roscoff, France(2)

Malaria is still a global health and economic burden, responsible for 2 million deaths annually. Plasmodium falciparum is the most pathogenic Plasmodium species causing severe clinical symptoms like cerebral malaria, renal failure and lactic acidosis. However, the less understood pathology and main cause of infant mortality in endemic countries is severe malarial anemia (SMA). SMA is accompanied by splenomegaly and macrophage hyperplasia. Therefore, the elucidation of the mechanism facilitating the massive accumulation of phagocytes in the spleen could give an insight into the severe pathology of malarial anemia.By applying confocal microscopy and live cell imaging, we visualized the recruitment of circulating monocytes to splenic macrophages, which had been challenged with P. falciparum infected red blood cells (iRBCs). We observed the accumulation of CCR2+ monocytes around RPMs upon iRBC erythrophagocytosis. Moreover, the recruited monocytes show a red pulp macrophage-like phenotype characterized by the upregulation of the RPM specific transcription factor, SpiC. This process is dependent on the direct cell interaction with splenic macrophages and the erythrophagocytosis of iRBCs. Together these findings indicate the recruitment and accumulation of monocytes to the spleen upon malaria infection, thus, contributing to macrophage hyperplasia and the subsequent massive destruction of uninfected and infected red blood cells, ultimately leading to severe malarial anemia.

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EMDS2018

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P30REPEATED MYCOBACTERIA VACCINATION IN MICE INDUCES MYELOID-DERIVED SUPPRESSOR CELL KILLING OF SPLENIC DENDRITIC CELLS VIA INOS-DEPENDENT NO PRODUCTION

Eliana Ribechini(1) - Ina Eckert(1) - Andreas Beilhack(2) - Nelita Du Plessis(3) - Gerhard Walzl(3) - Ulrike Schleicher(4) - Uwe Ritter(5) - Manfred B. Lutz(1)

Institute of Virology and Immunobiology, University of Würzburg, Würzburg(1) - Department of Internal Medicine Ii, University Hospital, Würzburg(2) - Division of Molecular Biology and Human Genetics, Stellenbosch University, Cape Town(3) - Institute of Microbiology, Friedrich-alexander-universität Erlangen-nürnberg, Erlangen(4) - Regensburg Center For Interventional Immunology, University of Regensburg, Regensburg(5)

Myeloid-derived suppressor cells (MDSCs) accumulate in patients with tuberculosis (TB) and a vaccine based on Mycobacterium tuberculosis (Mtb) is lacking. From this, we hypothesized that Mtb-based vaccines may induce MDSCs impairing vaccination success. Our data indicate that in vitro, bone marrow-derived resting MDSC (R-MDSC) stimulation with heat-killed Mtb resulted in the production of NO, directly suppressing T cells and inducing bone marrow-derived dendritic cell (BM-DC) apoptosis. The killing was NO dependent since blocking of iNOS reverted the effect. In vivo, repeated immunization of mice with Complete Freund´s Adjuvant (CFA) containing Mtb but not Incomplete Freund’s Adjuvant (IFA) lacking the Mtb component induced activated MDSCs (A-MDSCs) in the spleen. Myeloid cells isolated from spleens of CFA/CFA injected mice but not single CFA or CFA/IFA injected mice suppressed CD4+ and CD8+ T cell proliferation in a nitric oxide (NO) dependent manner. The accumulation of Gr-1+ CD11b+ iNOS+ myeloid cells was restricted to the splenic red pulp and bridging channels. Short term microbial challenge of mice in vivo induced infiltration of iNOS+ A-MDSCs after 6h into the white pulp resulting in conventional DCs (cDCs) and plasmacytoid DCs (pDCs) apoptosis in the T cell areas of the white pulp after 24h. DC apoptosis was not observed after microbial challenge alone and was reduced in NOS2-/- mice. In contrast, apoptosis of T cells was not observed and macrophage killing occurred but was independent of NO. Together, our data indicate that Mtb vaccines induced and activated MDSCs in spleens of mice leading to NO-dependent DC killing.

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EMDS2018

53

P31DENDRITIC CELLS PRE-CONDITION NAIVE T CELLS TO PROMOTE THEIR EFFECTOR DIFFERENTIATION POTENTIAL

Thomas Kreutzberg(1) - Caroline Wrangler(1) - Christine Schmitt-Mbamunyo(1) - Günter J. Hämmerling(2) - Natalio Garbi(1)

Institute of Experimental Immunology, University of Bonn, Bonn, Germany(1) - German Cancer Research Center, Dkfz, Heidelberg, Germany(2)

It is generally assumed that different naive T-cell clones have a similar potential to proliferate and differentiate into effector cells upon cognate recognition. However, it remains unresolved whether naive T-cells autonomously respond to cognate activation or whether integration of prior environmental cues shapes the magnitude and quality of antigen-specific T-cell responses. We found that naive T-cells exited quiescence with different kinetics independently of TCR specificity. Once entering the cell cycle, however, T-cells progressed through further divisions with similar speed, indicating that the time required from quiescence to first cell division fundamentally shapes the asynchronous characteristics of a dividing T-cell population. Environmental cues provided by self-recognition or interaction with dendritic cells prior to TCR stimulation contributed to this heterogeneity by accelerating naive T-cells exit from quiescence in response to subsequent cognate stimulation. Interestingly, naive T-cell pre-conditioning by DCs prior to cognate stimulation was critical not only for accelerated entry into the cell cycle, but also for acquisition of optimal effector Th1 program in terms of T-bet and IFNg expression. Dendritic cell loss during sepsis de-sensitised naive CD4 T cells resulting in loss of tonic TCR signalling, and decreased proliferation and differentiation in response to subsequent antigenic challenge, which may explain the observed impairment in Th1 differentiation obsrved in sepsis survivors. These results demonstrate that pre-conditioning of naive CD4 and CD8 T cells by DCs prior to cognate recognition is a key process shaping the magnitude and quality of the T-cell response.

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EMDS2018

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P32RELMα EXPRESSING MACROPHAGES PROVIDE SKIN BARRIER FUNCTION AND PROTECT FROM FATAL LUNG DAMAGE DURING HELMINTH INFECTION

Branislav Krljanac(1) - Christoph Koch(1) - Roland Naumann(2) - Stefan Wirtz(3) - Stephan Culemann(4) - Gerhard Krönke(4) - David Voehringer(1)

University Hospital Erlangen, Department of Infection Biology, Erlangen(1) - Mpi of Molecular Cell Biology and Genetics, Transgenic Core Facility, Dresden(2) - University Hospital Erlangen, Department of Medicine 1 - Gastroenterology, Erlangen(3) - University Hospital Erlangen, Department of Internal Medicine 3 - Rheumatology and Immunology, Erlangen(4)

M2-polarized macrophages (MΦs) can contribute to wound healing, regulation of glucose and fat metabolism, resolution of inflammation and protective immunity against helminths. Their differentiation, tissue distribution and effector functions are incompletely understood. Murine M2 MΦs express high levels of RELMα, an effector protein with potent immunomodulatory functions. To visualize RELMα+ MΦs in vivo and evaluate their role in defense against helminths we generated RELMαreporter/deleter mice. Infection with the helminth Nippostrongylus brasiliensis induced expansion of RELMα+ lung interstitial but not alveolar MΦs in a STAT6-dependent manner. Strikingly, RELMα+ MΦs were required for prevention of fatal lung damage during primary infection. Furthermore, protective immunity was lost upon specific deletion of RELMα+ MΦs by loss of larval retention in skin during secondary infection.Thus, RELMα reporter/deleter mice reveal compartmentalization of M2 MΦs in different tissues and demonstrate their critical role in resolution of severe lung inflammation and protection against migrating helminths.

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EMDS2018

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P33COMBINED PHOSPHOPROTEOME AND TRANSCRIPTOME ANALYSIS OF MACROPHAGES STIMULATED WITH MYCOBACTERIAL CORD FACTOR REVEALS A DICHOTOMY OF MINCLE-DEPENDENT AND -INDEPENDENT SIGNALING

Roland Lang(1) - Madlen Hansen(2) - Julian Peltier(3) - Barbara Killy(4) - Gernot Schabbauer(5)

University Hospital Erlangen, Institute of Clinical Microbiology, Immunology and Hygiene, Erlangen(1) - University Hospitel Erlangen, Institute of Clinical Microbiology, Immunology and Hygiene, Erlangen(2) - Newcastle University, Institute of Cellular and Molecular Biosciencess, Newcastle(3) - University Hospital, Institute of Clinical Microbiology, Immunology and Hygiene, Erlangen(4) - Medical University Vienna, Institute of Vascular Biology, Vienna(5)

Immune sensing of Mycobacterium tuberculosis relies on recognition by macrophages. Mycobacterial cord factor, trehalose-6,6’-dimycolate (TDM), is the most abundant cell wall glycolipid and binds to the C-type lectin receptor (CLR) MINCLE. To explore the kinase signaling linking the TDM-MINCLE interaction to gene expression, we employed quantitative phosphoproteome analysis. TDM caused upregulation of 6.7% and suppressed 3.8% of the 14,000 phospho-sites identified on 3727 proteins. MINCLE-dependent phosphorylation was observed for canonical players of CLR signaling (e.g. PLCg, PKCd), and was enriched for PKCd and GSK3 kinase motifs. MINCLE-dependent activation of the PI3K-AKT-GSK3 pathway contributed to inflammatory gene expression and required the PI3K regulatory subunit p85a. Unexpectedly, a substantial fraction of TDM-induced phosphorylation was MINCLE-independent, a finding paralleled by transcriptome data. Bioinformatic analysis of both datasets concurred in the requirement for MINCLE for innate immune response pathways and processes. In contrast, MINCLE-independent phosphorylation and transcriptome responses were linked to cell cycle regulation. Collectively, our global analyses show substantial reprogramming of macrophages by TDM and reveal a dichotomy of MINCLE-dependent and -independent signaling linked to distinct biological responses.

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EMDS2018

56

P34CELL-TYPE SPECIFIC EXPRESSION OF HFE DETERMINES THE COURSE OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM INFECTION IN MICE

Christoph Metzendorf(1) - Maja Vujic-Spasic(1) - Andrea Schroll(2) - David Haschka(2) - Alexander Hoffmann(2) - Richard Sparla(1) - Christian Huck(3) - Heribert Talasz(4) - Matthias Hentze(5) - Martina Muckenthaler(1) - Günter Weiss(2) - Manfred Nairz(2)

Department of Pediatric Hematology, University of Heidelberg, Heidelberg, Germany(1) - Internal Medicine Ii, Medical University Innsbruck, Innsbruck, Austria(2) - Institute For Analytical Chemistry and Radiochemistry, University of Innsbruck, Innsbruck, Austria(3) - Division of Clinical Biochemistry, Medical University Innsbruck, Innsbruck, Austria(4) - Molecular Medicine Partnership Unit, University of Heidelberg, Heidelberg, Germany(5)

Iron deposition in hepatocytes and iron depletion of the myeloid compartment are hallmarks of hemochromatosis type I, a hereditary disorder caused by mutations in HFE. Although HFE is expressed on macrophages, its functions in innate immunity remain incompletely understood.We herein compared putative immune-regulatory roles of Hfe in hepatocytes and myeloid cells, respectively, using a murine infection model with Salmonella Typhimurium, a macrophage-tropic bacterium with iron-dependent virulence. We found that total and macrophage-specific deletion of Hfe resulted in increased expression of nitric oxide synthase-2 in the spleen and protected mice from Salmonella infection. In contrast, mice with specific deletion of Hfe in hepatocytes succumbed earlier to Salmonella infection because of unrestricted extracellular bacterial growth associated with impaired generation of interleukin-6, interferon-γ and nitric oxide. Mice subjected to oral iron overload phenocopied the latter scenario suggesting that an increase in the serum iron pool, as found in hepatocyte specific Hfe knock-out mice, is deleterious in Salmonella infection.Based on these findings we conclude that tissue specific expression of Hfe contrastingly determines susceptibility to infection by divergently affecting iron availability for microbes and impacting on innate immune responses. We speculate that the lack of Hfe specifically in macrophages protects from infection with intracellular bacteria and contributed to the evolutionary conservation of human HFE mutations.

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EMDS2018

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P35NA+ BOOSTS ANTIBACTERIAL DEFENSE BY ENHANCING NFAT5-DEPENDENT AUTOPHAGOLYSOSOME FORMATION

Patrick Neubert(1) - Andrea Weichselbaum(1) - Carmen Reitinger(1) - Valentin Schatz(1) - Agnes Schröder(2) - John R. Ferdinand(3) - Anna-Lorena Bär(1) - Christoph Brochhausen(4) - Roman G. Gerlach(5) - Stefan Tomiuk(6) - Ger Van Zandbergen(7) - Katrina Binger(8) - Dominik N. Müller(9) - Kento Kitada(10) - Menna R. Clatworthy(3) - Christian Kurts(11) - Zeinab Abdullah(11) - Jonathan Jantsch(1)

Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg(1) - Institute of Orthodontics, University Hospital Regensburg, Regensburg(2) - Molecular Immunity Unit, University of Cambridge Department of Medicine, Cambridge(3) - Institute of Pathology, University Hospital Regensburg, Regensburg(4) - Project Group 5, Robert Koch Institute, Wernigerode(5) - Miltenyi Biotec Gmbh, Miltenyi Biotec Gmbh, Bergisch Gladbach(6) - Division of Immunology, Paul-ehrlich-institute, Langen (7) - Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville(8) - Experimental and Clinical Research Center, Max-delbrück Center and Charitè Berlin, Berlin(9) - Cardiovascular and Metabolic Disorders, Duke-nus Medical School, Singapore(10) - Institute of Experimental Immunology, University of Bonn, Bonn(11)

Infection and inflammation induce diet-independent Na+-accumulation without commensurate water retention in afflicted tissues, which favors the pro-inflammatory activation of macrophages and augmented their antibacterial and antiparasitic activity. While boosted antiparasitic control against Leishmania major is mediated by increasing the expression of the leishmanicidal type 2 NO synthase (NOS2), the molecular mechanisms underpinning this enhanced antibacterial defense with high Na+ (HS) exposure are unknown. Here, we provide evidence that HS-increased antimicrobial activity against E. coli was not dependent on NOS2 or on phagocyte oxidase. In contrast, HS-augmented antibacterial defense hinged on autophagolysosome formation and targeting of intracellular E. coli to acidic autophagolysosomal compartments in a ‘nuclear factor of activated T cells 5’ (NFAT5)-dependent manner. Altogether, these findings demonstrate that autophagolysosomal activity is a novel target of Na+-modulated macrophage function.

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EMDS2018

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P37MYELOID PTEN PROMOTES OBESITY-INDUCED INSULIN RESISTANCE

Julia Stefanie Brunner(1) - Andrea Vogel(1) - Alexander Lercher(2) - Mario Kuttke(1) - Omar Sharif(1) - Gernot Schabbauer(1)

Institute For Vascular Biology, Medical University Vienna, Vienna, Austria(1) - Cemm, Cemm, Vienna, Austria(2)

Modern lifestyle associated with excessive caloric intake and reduced physical exercise have led to a dramatic worldwide increase in the prevalence of obesity. Obesity is not only characterized by an exacerbated weight gain, but is also critically linked to an interconnected cluster of metabolic disorders and pathophysiologies including liver disease, arthritis, cancer and cardiovascular complications. During obesity, adipose tissue macrophages accumulate within adipose exerting a unique nutrient-triggered inflammatory phenotype. Therefore, they play a key role in obesity-induced chronic-low grade inflammation and insulin resistance. The Phosphatidylinositol 3-kinase (PI3K) plays a central role in orchestrating several intracellular processes, including proliferation, metabolism, survival and apoptosis. PI3K action is counteracted by protein phosphatases, among these PTEN. We and others could previously show that PTEN deficient myeloid cells exhibit a potent anti-inflammatory phenotype. We therefore hypothesized that myeloid PTEN deficient mice will display an improved outcome in the context of obesity induced insulin resistance.We subjected male PTENfl/fl LysMCre+/- (PTEN KO) and Cre negative littermate controls to a 16-week high fat diet regime. The in vivo insulin sensitivity was determined and the adipose tissue immune cell populations of these mice were characterized. Further, we performed various in vitro approaches, focusing on foam cell formation and cellular metabolism of PTEN deficient macrophages.We could show that myeloid PTEN deficiency, resulting in sustained PI3K activity led to beneficial effects following metabolic stress. Myeloid PTEN deficient mice exhibited improved insulin sensitivity and reduced adipose tissue inflammation post diet induced-obesity. In agreement, bone marrow transplantation of PTEN overexpressing immune cells was associated with elevated insulin resistance and dyslipidemia. Overall, our data provide new insights in how PI3K activity in myeloid cells shapes metabolic health during obesity, assigning a crucial role for myeloid PI3K activity that is independent of well-described effects of PI3K on insulin signaling.





EMDS2018

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P38INFLAMMATORY MONOCYTE-DERIVED MACROPHAGE WNT SIGNALLING REQUIRED FOR BRAIN REGENERATION

Claire Davies(1) - Anna Williams(2) - Veronique Miron(1)

University of Edinburgh, Queen’s Medical Research Institute, Edinburgh(1) - University of Edinburgh, Scottish Centre For Regenerative Medicine, Edinburgh(2)

Microglia and monocyte-derived macrophages are essential for driving myelin regeneration, termed remyelination, in the central nervous system. During myelin injury microglia and monocyte-derived macrophages have distinct functions, however the indiviudal contribution of these myeloid cell populations during remyelination is unknown. As remyelination is impaired in various neurological conditions, such as progressive multiple sclerosis, there is a pressing need to determine the mechanisms underlying this process in order to develop effective pro-remyelination therapies. Therefore, understanding the contribution of microglia and monocyte-derived macrophages during efficient remyelination will reveal targets that can be harnessed in order to drive remyelination. Here we investigated the role of microglia and monocyte-derived macrophages during remyelination by analysing their transcriptomes in vivo using RNA sequencing. We isolated microglia (CD11b+ CD45lo) and monocyte-derived macrophages (CD11b+ CD45hi) from focal demyelinated lesions of the adult mouse corpus callosum during remyelination. These data reveal that microglia and monocyte-derived macrophages have differential expression of genes and pathways at distinct stages of the regenerative process. Pathway analysis revealed the infiltrating monocyte-derived macrophages have enriched Wnt signalling, which has previously been associated with peripheral tissue regeneration.The monocyte-derived macrophages also had upregulation of genes assocaited with ECM maintenance and factors previously associated with remyelination.To further understand the role of infiltrating monocyte-derived macrophages during remyelination we compared transcriptomes of cells extracted from wildtype versus Ccr2-/- mice, in which inflammatory monocytes are excluded from the lesion site and do not remyelinate efficiently. Interestingly we recovered a CD11b+ CD45hi population from Ccr2-/- mice, suggestive of a different monocyte subset recruited independently of CCR2. The monocyte-derived macrophages present in Ccr2-/- lesions have a dysregulated Wnt signature and decreased expression of genes associated with remyelination. Thus, our data reveals a pro-regenerative monocyte-derived macrophage subset required for efficient remyelination, and identifies potential monocyte-derived macrophage signalling pathways that can be manipulated in order to enhance remyelination.





EMDS2018

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P39

A UNIQUE MACROPHAGE SUBSET THAT PLAYS A ROLE IN SARCOID GRANULOMA FORMATION

Clarice Lim(1) - Stefanie Hörer(2) - Julia Matschinger(2) - Karine Köhrer(3) - Thomas Krausgruber(4) - Thomas Weichhart(2)

Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria(1) - Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria(2) - Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical Universitcenter of Pathobiochemistry and Genetics, Institute of Medical Geneticsy of Vienna, Vienna, Austria(3) - Cemm Research Center for Molecular Medicine of The Austrian Academy of Sciences, Cemm, Vienna, Austria(4)

Sarcoidosis is a granulomatous, multi-organ disease primarily affecting the lungs of patients. Although asymptomatic at onset of disease, it can become difficult to treat, and present significant morbidity in one-third of patients as the disease progresses. We have previously shown that active mTORC1 signaling in granulomatous lesions of patients is associated with a progressive form of the disease, and that activation of mTORC1 by deletion of Tsc2 in macrophages was sufficient to initiate and maintain granulomas in mice. We were also able to show complete resolution of granulomas after treatment with mTOR inhibitor in mice, thus providing us with a novel mouse model of sarcoidosis. Here, we further characterize the myeloid cell populations in mice that develop sarcoid granulomas; and identify a special CD11c-expressing macrophage subset that arises and accumulates in these mice. We show here that this macrophage population is essential in initiating and maintaining granuloma formation. Additionally, our study also provides novel markers for identifying the main initiating cell population that aggregates during pulmonary sarcoidosis.





EMDS2018

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P40IMMUNOSUPPRESSIVE BONE-MARROW DERIVED MACROPHAGES ACCUMULATE IN THE CORE OF HUMAN GLIOBLASTOMAS

Laura Pinton(1) - Elena Masetto(1) - Marina Vettore(2) - Samantha Solito(2) - Sara Magri(2) - Marta D’Andolfi(2) - Paola Del Bianco(3) - Giovanna Lollo(4) - Alessandro Della Puppa(5) - Susanna Mandruzzato(6)

Istituto Oncologico Veneto, Immunologia Diagnostica Molecolare Oncologica, Padova, Italy(1) - University of Padova, Department of Surgery, Oncology and Gastroenterology, Padova, Italy(2) - Istituto Oncologico Veneto, Clinical Trials and Biostatistic Unit, Padova, Italy(3) - Lunam Universite, Univ Lyon, Université Claude Bernard Lyon 1, Lyon, France (4) - Azienda Ospedaliera Di Padova, Neurosurgery Unit, Padova, Italy(5) - Istituto Oncologico Veneto, University of Padova, Padova, Italy(6)

The complex interplay between cancer and immune system plays an essential role in tumor progression and its fine regulation determines patient outcome. In glioblastoma patients, a strong myeloid infiltrate is present at the tumor site, including populations with immunosuppressive properties, but a clear characterization of circulating and tumor-infiltrating myeloid cells still lacks in these patients. We studied the cell composition of glioblastoma biopsies and observed the presence of a leukocyte infiltrate mainly composed of macrophages, expressing the immunoregulatory molecule PD-L1, and a minority of T cells that show markers associated to impaired T cell function. By analyzing in detail the phenotype of macrophages present at the tumor site, we were able to distinguish between resident microglia (MG) and bone-marrow derived macrophages (BMDMs) and to study their localization and functional activity in the tumor lesion. In fact, preoperative administration of 5-aminolevulinic acid allows to identify and surgically remove different areas of the tumor through protoporphyrin IX emission of fluorescence. We observed that BMDMs accumulate in the center of the tumor, where they exert a strong immunosuppressive activity, while MG is the main population present in the margin and it is endowed with little or no suppressive capability. We also noticed alterations of the myeloid compartment in the peripheral blood of glioblastoma patients, with a significant decrease of intermediate monocyte population and an increase of CCR2+ cells among the same subset, suggesting that BMDM accumulation at tumor site could be sustained by an altered myelopoiesis in these patients.





EMDS2018

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P41DENDRITIC CELL PRODUCTION OF THE CHEMOKINE CCL17 IS REQUIRED FOR OPTIMAL TH2 RESPONSE INDUCTION AGAINST BOTH HELMINTHS AND ALLERGENS

Amanda Ridley(1) - A.T. Phythian Adams(2) - F.R. Svedberg(2) - D. Cunoosamy(3) - I. Förster(4) - E.M. Bignell(5) - M. Feeney(6) - P.C. Cook(2) - A.S. Macdonald(2)

Manchester Collaborative Centre For Inflammation Research (MCCIR), Univeristy of Manchester, Manchester, United Kingdom(1) - Manchester Collaborative Centre For Inflammation Research (MCCIR), University of Manchester, Manchester, United Kingdom(2) - Astrazeneca R&d, Respiration, Inflammation and Autoimmunity, Mölndal, Sweden(3) - Immunology and Environment, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany(4) - Manchester Fungal Infection Group (MRG), University of Manchester, Manchester, United Kingdom(5) - Glaxosmithkiine, C3 Dpu, Stevenage, United Kingdom(6)

Dendritic cells (DCs) are necessary and sufficient to induce type-2 immunity, which promotes protection against parasitic worm (helminth) infections whilst also mediating pathogenic allergic inflammation. However, the precise mechanisms that DCs employ to initiate and direct type-2 responses remain poorly understood. We previously showed that the methyl-CpG-binding domain protein, Mbd2, plays a major role in regulating DC gene expression and induction of type-2 immunity. We found that Mbd2 regulated CCL17 expression by DCs, suggesting that this Th2 associated chemokine may play a key role in governing the ability of DCs to promote Th2 responses against either allergens or helminths. Using Ccl17eGFP reporter mice, we have identified that DCs are an important cellular source for CCL17 during homeostasis and type-2 inflammation. We found that pulmonary CD11b-CD103+ and CD11b+MGL2+ DCs were the major DC subsets that express CCL17 in both naïve and house dust mite challenged mice. Further, DC expression of CCL17 was critical for their induction of optimal type-2 responses in vitro and in vivo, against either helminth antigens or allergens. Although Ccl17-deficient mice showed unimpaired Th2 responses following exposure to HDM, a CCR4 antagonist markedly reduced allergic lung inflammation; suggesting a potential requirement for CCL22 (which shares use of the receptor CCR4 with CCL17) in promoting Th2 allergic responses in the lung. Together these data emphasise the importance of both CCL17 and CCL22 for the optimal priming of type-2 immunity and highlights them as potential therapeutic targets during type-2 inflammatory diseases.





EMDS2018

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P42MIR-146A A KEY PLAYER IN BONE METABOLISM

Victoria Saferding(1) - Melanie Hofmann(2) - Julia S. Brunner(2) - Mihaela F. Militaru(1) - Antonia Puchner(1) - Silvia Hayer(1) - Gernot Schabbauer(2) - Melanie Timmen(3) - Richard Stange(3) - Josef S . Smolen(1) - Stephan Blueml(1)

Rheumatology, Medical University Vienna, Vienna(1) - Vascular Biology and Thrombosis Research, Medical University Vienna, Vienna(2) - Institute for Experimental Muskuloskeletal Medicine, University Hospital Muenster, Muenster(3)

Micro RNAs (miRNAs) play a crucial role in the regulation of bone metabolism. MiR-146a, an important anti-inflammatory miRNA, was found to negatively impact osteogenesis and bone regeneration in vitro, by controlling the differentiation of mesenchymal stem cells. But to date the role of miR-146a in bone metabolism and osteoporosis is not known.Systemic bone of wt and miR-146a deficient animals was assessed histologically and via µCT analysis, over 3 to 18 months of age. Serum cytokine levels were analysed by Elisa. MRNA expression levels in bone were analysed by qPCR. Ovariectomy (OVX) induced bone loss was performed. When we analysed bone volume of long bones histologically as well as with µCT analysis we detected significantly increased trabecular bone mass in miR-146a-/- compared to wt animals, starting at an age of 6 months. However, cortical thickness of systemic bones from miR-146a-/- animals was significantly reduced compared to control mice. Analysis of serum in aged miR-146a deficient animals displayed elevated activity of bone resorbing osteoclasts as amounts of CTX I in miR-146a-/- mice were significantly increased compared to wt animals. Q-PCR analysis of osteoclast as well as osteoblast marker genes in bones displayed elevated expression of signature molecules of both cell types in aged miR-146a-/- mice, suggesting a regulatory role of miR-146a in both. When we induced osteoporosis using the OVX disease model, histological analysis showed significant trabecular bone loss in wt mice. In contrast, we detected no trabecular bone loss in ovariectomized miR-146a-/- animals, suggesting that miR-146a deficiency protects bone loss induced by estrogen deficiency. MiR-146a seems to control bone turnover and miR-146a deficient mice accrue bone over time. This miRNA has a negative influence on bone loss occurring during oestrogen loss induced osteoporosis. Therefore miR-146a could be used as a therapeutic target in the treatment of osteoporosis.





EMDS2018

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P43NA+ INDUCED NFAT5 AS A NOVEL REGULATOR OF OSTEOCLASTOGENESIS

Agnes Schröder(1) - Patrick Neubert(2) - Jens Titze(3) - Aline Bozec(4) - Wolfgang Neuhofer(5) - Peter Proff(1) - Christian Kirschneck(1) - Jonathan Jantsch(2)

Department of Orthodontics, University Hospital Regensburg, Regensburg, Germany(1) - Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany(2) - Duke-national University of Singapore, Duke-national University of Singapore, Singapore, Singapore(3) - Department of Internal Medicine 3, Friedrich-Alexander-University Erlangen-nürnberg, Erlangen, Germany(4) - Department of Nephrology, Helios Klinikum Erfurt, Erfurt, Germany(5)

Dietary salt consumption induces cutaneous Na+ accumulation and is linked with various diseases like hypertension, autoimmune diseases and osteopenia. Increases in local Na+ levels trigger an osmoprotective response in macrophages that favours proinflammatory macrophage activation and curtails their regulatory activity. However, the impact of Na+-rich diets on osteoclastogenesis is unexplored.Here, we observed that, in comparison to high Na+ diets (HSD), low Na+ diets (LSD) increased bone density and augmented the bone marrow (BM) Na+ stores by 40 mmol/g to 180 mmol/g. This was paralleled by increases in the expression of the osmoprotective transcription factor nuclear factor of activated T cell 5 (NFAT5), in the bone marrow. This demonstrates that the local Na+-environment in the BM of animals exposed to LSD is best mimicked by high Na+ cell culture conditions (HS¢), while HSD conditions are best simulated by exposing cells to normal (standard) cell culture condition (NS¢).HS¢ impaired RANK-L (receptor activator of NF-kB ligand)-triggered osteoclastogenesis in a NFAT5-dependent manner. In line with this, mice with conditional Nfat5-deficiency in the myeloid cell compartment displayed reduced bone density. This work provides new insights into mechanisms of Na+-induced cessation of osteoclastogenesis and offers new targets for treating salt-induced osteopenia.





EMDS2018

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P44SERA OF PATIENTS WITH AXIAL SPONDYLOARTHRITIS (AXSPA) ENHANCE OSTEOCLASTOGENIC POTENTIAL OF MONOCYTES ISOLATED FROM HEALTHY INDIVIDUALS

Mariusz Korkosz(1) - Marcin Czepiel(2) - Zofia Gula(1) - Malgorzata Stec(2) - Kazimierz Weglarczyk(2) - Magdalena Rutkowska-Zapala(2) - Anna Gruca(2) - Marzena Lenart(2) - Jaroslaw Baran(2) - Jerzy Gasowski(3) - Przemyslaw Blyszczuk(2) - Maciej Siedlar(2)

Jagiellonian University Medical College, Department of Rheumatology, Krakow, Poland(1) - Institute of Pediatrics, Jagiellonian University Medical College, Department of Clinical Immunology, Krakow, Poland(2) - Jagiellonian University Medical College, Department of Internal Medicine and Gerontology, Krakow, Poland(3)

BackgroundAxial spondyloarthropathy (axSpA) is characterized by significant bone loss caused by dysregulation of physiological bone turnover, possibly resulting from intensified differentiation of osteoclasts. The aim of this study was to reevaluate the levels of osteoclastogenesis-mediating factors: soluble RANKL, M-CSF, OPG and other cytokines in sera of untreated axSpA patients and to test whether they influence differentiation of healthy monocytes towards osteoclasts.

MethodsMonocytes of healthy individuals were cultured in vitro in presence of sera from randomly chosen axSpA patients and controls, with addition of exogenous M-CSF and/or RANKL or without. Osteoclastic differentiation was assessed analyzing osteoclast markers (cathepsin K- and RANK- mRNAs) and with osteoclast-specific staining.

ResultsAxSpA patients’ sera levels of soluble RANKL were significantly lower and MCSF, IL-6, OSM, IL-17A and TNFα significantly higher in comparison to controls. Numbers of generated osteoclasts and cathepsin K-mRNA levels did not differ between cultures supplemented with sera of healthy and axSpA patients, both in the absence/presence of M-CSF. Instead, addition of exogenous RANKL boosted osteoclastogenesis, which was significantly higher in cultures with axSpA sera. Sera from axSpA patients induced RANK-mRNA, independently of M-CSF and RANKL stimulation.

ConclusionParadoxically, serum levels of soluble RANKL observed in axSpA are significantly lower in comparison to controls. This indicate that sera of axSpA patients - in contrary to healthy subjects – contain soluble factors able to stimulate healthy monocytes responsiveness to relative low RANKL serum levels, by inducing high RANK-mRNA expression and - as a net effect - boosting their osteoclastogenic potential. This study was supported by grant from the National Science Centre (NCN) in Poland no. 2013/09/B/NZ6/02545 and Jagiellonian University Medical College grant K/ZDS/006120.





EMDS2018

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P45CHROMATIN STATE DYNAMICS AND GENE REGULATION IN MACROPHAGES DURING SKELETAL MUSCLE REGENERATION UPON AN ACUTE STERILE INJURY

Petros Tzerpos(1) - Andreas Patsalos(1) - Gergely Nagy(1) - Nikolas Giannakis(1) - Laszlo Holasz(1) - Balazs Dezso(1) - Laszlo Nagy(2)

Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary(1) - Institute For Fundamental Biomedical Research, John Hopkins All Children’s Hospital, St Petersburg, Fl, United States(2)

Skeletal muscle regeneration upon acute sterile injury is a dynamic process that is highly dependent on an in situ transition of muscle infiltrating blood monocytes to an inflammatory (Ly6C high) and then to a reparative (Ly6C low) macrophage phenotype. These diverse phenotypes are a combination of differentiation and polarization transcriptional programs that can be transient, stable or permanent, corresponding to cellular activation, polarization and differentiation states, respectively. Here, in order to delineate the temporal order of such transcriptional events we used the cardiotoxin (CTX) acute muscle injury and we systematically profiled chromatin openness (ATAC-seq) and gene expression (RNA-seq) in macrophage populations from injured muscles during the time-course of tissue repair/regeneration. We found a large class (>9.000) of genomic regulatory elements becoming transiently accessible during the first phase of the regeneration process, mainly near genes involved in tissue repair such as cytokines, chemokines, growth factors and stress response mediators. These de novo accessible regulatory sites were found to be highly enriched (>60%) for the AP-1 and MARE (AP-1 motif variation) motifs compared to known macrophage lineage factors’ specific motifs. We further identified BACH1, a MARE-binding and heme-regulated transcriptional repressor, as a potential regulatory molecule of macrophage differentiation and polarization. Genetic ablation of Bach1 severely affected inflammatory transcriptional programs and muscle tissue regeneration kinetics upon CTX injury. In addition, cistrome analysis (ChIP-seq) in bone marrow-derived macrophages revealed that BACH1 binds extensively to chromatin (>45.000 sites), mainly in active and poised enhancers but also to a large class of closed and inactive chromatin regions, suggestive of a bookmarking mode of action. Our data suggest that fine-tuning of transient inflammatory transcriptional programs in macrophages during tissue injury largely depend on AP-1/MARE motif binding transcription factors and that BACH1 is a major regulator of activation and resolution of the inflammatory response.





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P46LUNG DENDRITIC CELLS AS KEY MEDIATORS LINKING CIGARETTE SMOKE AND LUNG DISEASE DEVELOPMENT IN RHEUMATOID ARTHRITIS

Robert Vassallo(1)

Mayo Clinic, Department of Medicine, Rochester (1)

Epidemiological studies implicate cigarette smoking (CS) as a major risk factor for the development of autoimmune diseases like seropositive rheumatoid arthritis (RA). The mechanisms by which CS promotes autoimmunity – including RA – remain largely unknown, but likely involve modulation of dendritic cell (DC) functions. Because a significant proportion of RA patients develop lung disease, we postulated that direct effects of CS on lung myeloid DCs would result in an inflammatory response that promotes the generation of autoimmunity in the lung. To explore directly the effects of CS on lung and systemic DCs, C57B6 mice were placed in a TE-2 smoking chamber and chronically exposed to CS generated from Kentucky research cigarettes. Murine DCs were extracted from the spleen, lungs and lymph nodes at the time of sacrifice, and analyzed for viability, T cell proliferative potential, and cytokine/chemokine secretion. DCs extracted from mice exposed to ≥4 weeks of CS exposure demonstrated enhanced viability and survival compared to control DCs. The levels of certain co-stimulatory molecules were enhanced on DCs extracted from CS exposed mice compared to wild type mice. DC-mediated proliferative T cell responses induced by allogeneic DCs were not significantly different between CS exposed and control wild type mouse DCs. When activated by lipopolysaccharide (10ng/ml), DC extracted from CS exposed mice produced lower amounts of the Th-1 polarizing cytokine IL-12p70: however, the production of TNFα, neutrophilic chemokines, and PGE2 levels were significantly higher by lung DCs extracted from CS exposed mice. These data indicate that the effect of CS on host DC function in vivo is complex: stimulatory, suppressive, as well as neutral responses occur. Determination of specific in vivo effects of CS on DC functions in the context of autoimmunity are essential to enable further investigation into specific mechanisms by which smoking promotes autoimmune diseases like RA.





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P47THE ROLE OF PI3K/PTEN IN MICROGLIA FUNCTIONS DURING HOMEOSTASIS AND NEUROINFLAMMATION

Andrea Vogel(1) - Melanie Hofmann(1) - Julia S. Brunner(1) - Martina Kerndl(1) - Hannes Datler(1) - Omar Sharif(1) - Gernot Schabbauer(1)

Institute For Vascular Biology and Thrombosis Research, Medical University Vienna, Vienna, Austria(1)

Microglia, the resident tissue macrophages of the central nervous system (CNS) are crucially involved in brain development, pathogen recognition and tissue homeostasis. Although, microglial activation is an extensively-described feature of many CNS diseases including multiple sclerosis, their exact role and impact on disease pathogenesis remains ill-defined. The Phosphatidylinositol 3-kinase (PI3K) is a central cellular hub and its signaling is negatively regulated by PTEN. Our previous published observations indicate that myeloid PTEN deficiency skews the inflammatory phenotype of macrophages. We therefore hypothesized that PI3K/PTEN might influence microglial functions during homeostasis and inflammation. Examining microglia from adult mice overexpressing or lacking myeloid specific PTEN expression (PTENTg/+ and PTENfl/fl

LysMCre/+ respectively) in steady state and during experimental autoimmune encephalomyelitis (EAE), revealed influences on microglial numbers and activation as defined using MHCII expression. Beside activated and resting microglia, EAE bearing mice further exhibit a population of infiltrating monocytes. Myelin scavenging, representing a crucial process in the resolution of autoimmune-driven CNS inflammation, was reduced upon PI3K inhibition in vitro. Our data thus provide new insights in how PI3K activity shapes CNS immunity during health and disease.





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P48AN INTEGRATIVE ANALYSIS OF TRANSCRIPTOMICS AND LIPIDOMICS DATA FROM HUMAN CD14+ MONOCYTES

Ioanna Gemünd(1) - Marie Oestreich(1) - Lea Seep(1) - Anna-Lena Hardt(1) - Thomas Ulas(1) - Christoph Thiele(1) - Joachim L. Schultze(1) - Anna C. Aschenbrenner(1)

Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany(1)

The use of transcriptomics to support biological analyses has increased tremendously in the last years. In immunology, it has been of great help to identify, distinguish and functionally describe cell populations and subsets as well as to determine differences in various disease contexts. Lipidomics, however, has not been used to similar extent for these questions – albeit the fact that in recent years, elevated levels of nutrients (such as cholesterol, triglycerides and free fatty acids), that are often contained in disproportionate amounts in contemporary diet, have been shown to represent anthropogenic stimuli implicated to cause low-level inflammation, termed ‘metaflammation’, and cue metabolic disorders. A crucial constituent in the development and progression of this pathology are cells of the innate immune system, particularly the myeloid compartment. Previous analyses by our lab had provided initial insights into changes in the transcriptional signatures of myeloid cells following exposure to free fatty acids. Depending on the nature of the fatty acid, the cells were specifically reprogrammed exemplifying their ability to react to such anthropogenic stimuli.As lipids can act as important signaling mediators aside from their structural function in membranes or metabolic role in energy storage, we now used advanced mass spectrometry-based assessment of lipid species to integrate lipidomics into our analyses by analyzing transcriptomes and lipidomes of human CD14+ monocytes after exposure to saturated or unsaturated fatty acids. Transcriptional signatures were screened for differences in metabolic pathways and overlaid with the observed changes in various lipid species. Lipidomics data was analyzed by k-means clustering of group fold change patterns for similarly regulated lipid species and screened for specific modules for the different experimental conditions. Transcriptional changes of key lipid metabolism components were reflected in the respective lipidomes. Integrating the assessment of transcriptomic and lipidomic profiles provides a powerful workflow for generating a more complete understanding of the general biological and metabolic processes in immune cells in various contexts. In the future, this approach will help study pathophysiological conditions and thus gain insight into molecular mechanisms of disease.





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P49TOLERANCE INDUCED BY IL-35IG-EXPRESSING DENDRITIC CELLS IS MEDIATED BY ARGINASE 1

Maria Laura Belladonna(1) - Eleonora Panfili(1) - Giada Mondanelli(1) - Claudia Volpi(1) - Ciriana Orabona(1) - Ursula Grohmann(1)

Istituzione, Department of Experimental Medicine, University of Perugia, Perugia, Italy(1)

Dendritic cells (DCs) are specialized antigen-presenting cells capable of triggering either stimulatory or regulatory T-cell immune responses, according to their cell maturation state and microenvironmental stimuli. IL-35, a heterodimeric cytokine belonging to the IL-12 family, is produced by regulatory T and B cells, and is known to induce a strong immunosuppressive response. Indoleamine 2,3-dioxigenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, degrading essential aminoacids and producing suppressive catabolites, importantly contribute to immunoregulation. Aiming to identify a possible link between IL-35 and these two enzymes, we first developed an in vivo model of negative vaccination to verify the suppressive action of ectopic single chain IL-35Ig produced by tolerizing DCs (DC35), and then we analyzed IL-35Ig effects on IDO1 and Arg1 enzymes. In this study, we observed that antigen presentation by DC35 induces a state of tolerance, which is not only long lasting, but also antigen-specific. The suppressive action of ectopic IL-35Ig produced by tolerizing DC35 triggers IDO1 activity in vivo in splenic DCs. Tolerance evocated by IL-35Ig is likely related to, but not dependent on IDO1 activity, since the transfer of Ido1-/- DC35 maintain a suppressive potential, too. Nevertheless, contrary to Ido1, Arg1 is induced in DC35, an observation suggesting a possible correlation between Arg1 activity and IL-35Ig tolerogenic effect. In fact, in a delayed-type hypersensitivity assay, treatment with the Arg1 inhibitor nor-NOHA before in vivo sensitization restores DC35 antigen presentation ability. Therefore, the observed Arg1, but not Ido1, induction in vitro by autocrine/paracrine IL-35Ig appears to be an early mechanism downstream IL-35, later involving IDO1 in the complex in vivo network of immune responses.





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P50EFFECT OF THE AIR POLLUTION – TRANSITION METAL CONTAINING PARTICULATE MATTER ON THE ACTIVITY OF HUMAN MONOCYTES

Adrianna Galuszka(1) - Malgorzata Stec(1) - Anna Gruca(1) - Maciej Siedlar(1) - Jarek Baran(1)

Institute of Pediatrics, Jagiellonian University Medical College, Department of Clinical Immunology, Cracow, Poland(1)

In recent years, an increase in air pollution exerted a serious impact on human health. A number of studies suggest that transition metal containing particulate matter (TMCPM) may play a role in the initiation and development of many disorders, including autoimmune diseases. In this context, we investigated the effect of TMCPM on antigen presenting capacity and pro-inflammatory response of peripheral blood monocytes.Monocytes were obtained from peripheral blood mononuclear cells by countercurrent centrifugal elutriation and cultured in vitro in RPMI 1640 medium supplemented with 10% FBS with or without TMCPM at three concentrations (1 µg/ml, 10 µg/ml, 100 µg/ml). Two different preparations of TMCPM were used: NIST (SRM 1648a- standard urban particulate matter obtained from US National Institute for Standards and Technology) and LAP (NIST 1648a treated with a low-temperature plasma). Antigen presenting capacity of monocytes, production of reactive oxygen species (ROS) and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF), mitochondrial membrane potential and Caspase-1 activation were examined after exposure to TMCPM. Reduced antigen presenting capacity to T lymphocytes and significant changes in cell morphology have been observed after exposure of monocytes to TMCPM. These effects were associated with an increased production of ROS, altered mitochondrial membrane potential and reduced viability of monocytes, occurring independently of the concentration or preparation of TMCPM that was used. Moreover, the activation of Caspase-1, as early as after 15 minutes of monocytes exposure to TMCPM followed by IL-1β production, was detected, suggesting the involvement of inflammasome in this process.TMCPM induce strong inflammatory response of monocytes suggesting their role in pathologies caused by air pollutions.This study was supported by the National Science Centre (NCN) of Poland, grant no. 2015/16/WST5/00005.





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P51A NOVEL KYNURENINE-DEPENDENT CIRCUIT IN DC1 PROMOTE IDO1 EXPRESSION IN DC2 LEADING TO EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS SUPPRESSION

Marco Gargaro(1) - Giulia Scalisi(1) - Carlos G. Briseño(2) - Giorgia Manni(1) - Vivek Durai(2) - Prachi Bagadia(2) - Derek J. Theisen(2) - Dario Nardi(1) - Paolo Puccetti(1) - Theresa L. Murphy(2) - Kenneth M. Murphy(2) - Francesca Fallarino(1)

University of Perugia, Department of Experimental Medicine, Perugia, Italy(1) - Washington University in St. Louis, School of Medicine, Department of Pathology and Immunology, St. Louis, United States(2)

Classical dendritic cells (cDCs) are professional antigen-presenting cells that play a key role in shaping appropriate immune responses. DCs are potent T cell activators but they are also involved in maintaining immune homeostasis and self-tolerance. DCs can be classified into three major types: pDC, DC1 and DC2. One mechanism by which DCs regulate tolerance involves indoleamine 2,3-dioxygenase 1 (IDO1) a tryptophan (Trp) metabolizing enzyme. In this study, we analyzed the ability of l-Kyn to induce tolerogenic IDO1 pathway in different DCs subsets in vitro and in vivo model of experimental autoimmune encephalomyelitis (EAE). We show that inflammatory stimuli, like LPS, was able to induce IDO1 only in DC1, but not in DC2 or pDC, when DCs were treated as isolated cultures. In contrast, when LPS was added to cultures containing all three DC subsets, LPS could also induce IDO1 expression in DC2, which acquired tolerogenic function. Induction of IDO1 in DC2 involved a novel DC1-DC2 communication pathway mediated by a Kyn-AhR-RelB axis. Kynurenine produced by DC1 activates AhR in DC2 inducing IDO1 in a RelB-dependent manner. In vitro l-Kyn treatment impaired DC2 T cells priming ability causing suppression of MOG-specific reactivity with an increment of Foxp3+ CD4+ T cells. In vivo, oral administration of l-Kyn induces functional Treg cells that suppress EAE and this effect is completely abrogated in Ahrflox/flox CD11C Cre+ mice. These data suggest that in specific microenvironments, small numbers of IDO1-expressing DC1 may spread tolerogenic activity to DC2 cells through a kynurenine-AhR axis and l-Kyn could constituting a unique endogenous molecule for therapeutic immunomodulation of inflammatory and autoimmune diseases.





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P52INDOLEAMINE 2,3-DIOXYGENASE 1 (IDO1): RELATIONSHIP BETWEEN FUNCTIONS AND INTRACELLULAR LOCALIZATION IN PLASMACYTOID DENDRITIC CELLS

Alberta Iacono(1) - Andrea Pompa(2) - Francesca De Marchis(2) - Michele Bellucci(2) - Fabio Grassi(3) - Elisa Albini(1) - Ursula Grohmann(1) - Maria Teresa Pallotta(1)

University of Perugia, Experimental Medicine, Perugia, Italy(1) - Institute of Biosciences and Bioresources of Perugia, Italian National Council of Research, Perugia, Italy(2) - University of Milano, Medical Biotechnology and Traslational Medicine, Milano, Italy(3)

Knowledge of protein spatial dynamics at the subcellular level is key to understanding protein functions in health and disease. Indoleamine 2,3-dioxygenase (IDO1), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions. We recently demonstrated that IDO1 does not merely degrade tryptophan and produce immunoregulatory kynurenines, but it also acts as a signal-transducing molecule, independently of its enzymic function. In particular, in a microenvironment dominated by the cytokine TGF-β, IDO1 is involved in intracellular signaling events responsible for self-amplification and maintenance of a stably regulatory phenotype in plasmacytoid dendritic cells (pDCs), a rare yet functionally flexible DC subset. Although the cytosolic localization of the IDO1 enzyme has been clearly established, the subcellular compartment of IDO1 as a signaling molecule has been unclear. By means of confocal microscopy and isopycnic gradient experiments, we here investigated the intracellular localization of IDO1 and its possible co-localization with subcellular structures. We detected the presence of a significant amount of IDO1 in structures other than cytoplasm, i.e., early endosomes, suggesting that IDO1 has not exclusively a cytoplasmic localization. This co-localization was more evident after treatment of pDCs with TGF-β. Thanks to these data, it is possible to assume the enzyme IDO1 has not exclusively a cytoplasmic localization, but there could be a balance between its localization in the cytosol and early endosomes, possibly related to the two distinct IDO1-mediated (catalysis vs. signaling) tolerogenic mechanisms.





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P53HYPERGLYCAEMIC CONTROL OF HISTONE CODE IN METABOLIC INFLAMMATION

Marije Mossel(1) - Kondaiah Moganti(1) - Harald Klüter(1) - Martin Harmsen(2) - Julia Kzhyshkowska(1)

University of Heidelberg, Medical Faculty Mannheim, Institute of Transfusion Medicine and Immunology, Mannheim, Germany(1) - Dept. Pathology and Medical Biology, University Medical Centre Groningen, Groningen, Netherlands(2)

Epigenetic mechanisms regulate development of diabetic vascular complications and metabolic memory. Histone code is an essential mechanism of epigenetic memory affected by diabetic conditions. Monocytes (Mo) and macrophages (MF) are key innate immune cells that control inflammatory reactions leading to vascular complications. However, the mechanisms of hyperglycemia-induced epigenetic changes in human macrophages are not known. Using Affymetrix microarray profiling and RT-PCR we identified that hyperglycaemia induces elevated gene expression of L-1beta, CCR2, S100A9 and S100A12 in primary human macrophages. Association for activating and repressing histone marks with the promoters of these genes was analyzed by chromatin immunoprecipitation. Hyperglycaemia induced the increased association of only activating H3K4me1, H3K4me3 but not repressive H3K9me3 and H3K27me3 histone marks with promoters of the selected genes in human M1 macrophages. Increased association of the activating histone marks correlated with induction of gene transcription. Application of inhibitors for histone methyltransferases showed that SET7/9 regulate expression levels of CCR2 and S100 proteins but not of IL1-beta, whereas SMYD3 specifically regulates S100A12. An inhibitor of MLL core complex methyltransferase activity had no effect on the expression levels of IL-1beta, CCR2, S100A9 and S100A12. We concluded that hyperglycaemia induces upregulation of pro-inflammatory factors in M1 macrophages by activating histone code on the gene promoters while gene-specific involvement whereas SMYD3 and SET7/9 are involved in induction of the specific gene transcription. Mechanism of action of the distinct methyltransferases in hyperglycaemic conditions remains to be identified and will add an insight in the role of hyperglycemia-induced inflammatory processes that lead to vascular complications in diabetes.





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P54N-ACETYLSEROTONIN RESTRAINS NEUROINFLAMMATION IN AN IDO1-MEDIATED FASHION IN DCS

Eleonora Panfili(1) - Alessandra Scarponi(1) - Claudia Volpi(1) - Antonio Macchiarulo(2) - Laura Santambrogio(3) - Ursula Grohmann(1)

University of Perugia, Department of Experimental Medicine, Perugia, Italy(1) - University of Perugia, Department of Pharmaceutical Sciences, Perugia, Italy(2) - Albert Einstein College of Medicine, Department of Pathology, New York, United States(3)

N-acetylserotonin (NAS) is a Trp metabolite generated by the arylalkylamine N-acetyltransferase (AANAT) enzyme from serotonin along the serotonin pathway. NAS is endowed with significant antidepressant, anti-ischemic and anti-oxidant actions in mice.Indoleamine 2,3-dioxygenase 1 (IDO1) is a Trp degrading enzyme which catalyzes the first and rate-limiting step in the kynurenine pathway, leading to Trp depletion and the production of a series of immunoregulatory molecules collectively known as kynurenines. Both effects are involved in the anti-inflammatory and immunoregulatory action of the enzyme by virtue of which it may represent a major therapeutic target in multiple sclerosis (MS). In fact, administration of 1-MT, the standard inhibitor of IDO1 catalytic activity, exacerbates the clinical course of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Because recently has been shown that NAS exerts protective effects in EAE induced by the myelin oligodendrocyte glycoprotein (MOG), we investigated whether IDO1 could be involved in NAS effects in this experimental model. We observed that NAS protective effects in EAE are lost in Ido1−/− mice. Since the highest IDO1 expression and catalytic activity occur in dendritic cells (DCs), the most potent antigen presenting cells, we next investigated whether DCs could represent the cell target of NAS effects. In a delayed-type hypersensitivity assay, treatment with NAS before in vivo sensitization causes, only in IDO1-competent mice, a suppression of DCs antigen presentation ability demonstrating that NAS, via IDO1, confers immunosuppressive effects on DCs.Therefore, we proved an in vivo IDO1 critical, DC mediated role in NAS neuroprotective effects contributing to dissect part of the mechanism underlying this Trp metabolite action.





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P55MYELOID DYSFUNCTION IN AN INDUCIBLE MOUSE MODEL OF GLYCOGEN STORAGE DISEASE TYPE 1B

Federica Raggi(1) - Anna Livia Pissavino(2) - Roberta Resaz(2) - Daniela Segalerba(2) - Andrea Puglisi(2) - Francesca Antonini(3) - Genny Del Zotto(3) - Alessandra Gamberucci(4) - Paola Marcolongo(4) - Luca Mastracci(5) - Federica Grillo(5) - Maria Carla Bosco(2) - Luigi Varesio(2) - Alessandra Eva(2)

Istituto G. Gaslini, Laboratory of Molecular Biology, Department of Translational Research, Genova, Italy(1) - Istituto G.gaslini, Laboratory of Molecular Biology, Department of Translational Research, Genova, Italy(2) - Istituto G.gaslini, Core Facilities Laboratory, Department of Translational Research, Genova, Italy(3) - University of Siena, Department of Molecular and Developmental Medicine, Siena, Italy(4) - University of Genova, Pathology Unit, Department of Surgical Sciences and Integrates Diagnostics, Genova, Italy(5)

Background Neutrophils and macrophages play a key role in host protection against microbial infections. Myeloid dysfunction is a common feature of many diseases and is observed in patients affected by Glycogen Storage Disease type 1b (GSD1b), in addition to the disrupted glucose homeostasis. GSD1b is a rare genetic autosomal recessive disease caused by the glucose-6-phospate transporter (G6PT) defect.The objective of this study was to understand the pathophysiology of the disease focusing on neutrophil and macrophage functional activity in an inducible KO mouse model.

MethodsWe used the conditional G6PT-deficient mouse to develop an inducible G6PT-/- model to allow temporally-regulated G6PT deletion by administration of tamoxifen (TM).Hystology, histochemistry, and phenotype analysis was performed at different time points after TM-induced G6PT inactivation. Myeloid cells were isolated and analyzed for functional activity.

ResultsTM-G6PT-/-mice showed metabolic abnormalities typical of disturbed glucose homeostasis and tissue damage characteristic of the human disease. Moreover, G6PT excision affects the energy homeostasis and functionality of myeloid cells leading to defects in respiratory burst, defective calcium signaling, impaired chemotaxis and phagocytic activity. We showed reduced hematopoiesis in response to G-CSF and M-CSF, bone marrow hypocellularity, and high rate of cell death in TM-G6PT-/-. Therefore, these mice manifest pathological symptoms similar to those of human GSD1b.

Discussion We have developed an inducible murine model that mimics the GSD1b pathophysiology in terms of tissues damage, myeloid dysfunction, and susceptibility to bacterial infection. Due to their long term survival, these animals may represent a valuable model to study the age-related disease progression and to discover biomarkers and new treatment interventions.





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P56THE IMPACT OF THE PGD2-DP1-DP2 AXIS IN RHEUMATOID ARTHRITIS

David Roula(1) - Kathrin Rohrer(1) - Martin Stradner(2) - Eva Sturm(1) - Akos Heinemann(1)

Otto Loewi Research Center, Pharmacology Section, Medical University of Graz, Graz(1) - Division of Rheumatology and Immunology, Medical University of Graz, Graz(2)

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting the joints with an incidence of 0.5 to 1% of the general population. Disease onset is caused by activation of local cell populations that attract immune cells to the synovial membrane, causing synovitis. The most influential cells are macrophages which produce a range of pro-inflammatory cytokines, resulting subsequently in cartilage degradation and bone erosion. It is hypothesized that one of these mediators is Prostaglandin (PG)D2, as it is elevated in the synovial fluid and blood of patients affected by RA. PGD2 acts on two GPCRs called DP1 and DP2, which are expressed in cells involved in RA pathophysiology, including monocytes, macrophages, dendritic cells, synovial fibroblasts and to some extent on neutrophils. Our interest is to observe whether the expression of both receptors is altered between acute patients, patients in remission and healthy controls using flow cytometry and fluorescence microscopy. In circulation we show that basophils display a significant reduction in DP2 expression in RA patients compared to healthy controls. In contrast, DP2 expression is upregulated in differentiated anti-inflammatory macrophages of acute patients compared to healthy controls and patients in remission. Monocytes within the PBMC fraction show no chemotactic activity in response to PGD2 or 5-OxoETE. ROS production and cytoskeletal rearrangement in granulocytes is not increased in RA patients in response to the same mediators. Synovial fibroblasts express DP1, DP2 and hPGDS, but no difference between controls and patients could be observed.We conclude that although rheumatoid arthritis displays systemic features, leukocytes in circulation are not heavily impacted by this disease in regard to the PGD2-DP axis. Anti-inflammatory macrophages however show an increase in DP2 expression in acute patients, suggesting a potential regulation via prostanoid metabolism.





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P57N-ACETYLSEROTONIN EXERTS NEUROPROTECTIVE EFFECTS ENHANCING IDO1 CATALYTIC ACTIVITY

Alessandra Scarponi(1) - Eleonora Panfili(1) - Claudia Volpi(1) - Antonio Macchiarulo(2) - Laura Santambrogio(3) - Ursula Grohmann(1)

University of Perugia, Department of Experimental Medicine, Perugia, Italy(1) - University of Perugia, Department of Pharmaceutical Sciences, Perugia, Italy(2) - Albert Einstein College of Medicine, Department of Pathology, New York, United States(3)

L-tryptophan, the least abundant essential amino acid, is the precursor of many metabolites involved in immune response control produced by the Kynurenine and Serotonin pathways. Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme containing enzyme which catalyze the first, rate limiting step in tryptophan catabolism via the first one. Both depletion of the amino acid and formation of kynurenines lead to tolerogenic effects. In the Serotonin pathway, N-acetylserotonin (NAS), first considered only as an intermediate metabolite, more recently has been shown to be effective in controlling neuroinflammation in experimental autoimmune encephalomyelites (EAE), a model for human multiple sclerosis. Starting from the evidence that NAS neuroprotective properties are lost in mice lacking IDO1 expression, we investigated the relationship between the Trp metabolite and the enzyme. We found that NAS acts as catalytic enhancer of IDO1 binding directly at an allosteric site of the enzyme, thus increasing the Kyn production in purified mouse cDCs and leading to an immunosuppressive response. Moreover, we demonstrated that NAS does not modulate IDO1 transcript and protein levels and is not able to protect the enzyme from proteasome degradation triggered by IL-6 in mice cDCs.In a scenario where IDO1 enzymatic enhancers have not been developed yet, NAS may represent an important natural therapeutic agent that could be used in autoimmune and neuroinflammatory disorders.





EMDS2018

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P58HYPERGLYCEMIA MODULATES INFLAMMATORY RESPONSES TO METAL NANOPARTICLES IN HUMAN MACROPHAGES

Tatjana Sevastyanova(1,2) - Alexandru Gudima(1) - Vladimir Ryabov(1,2) - Nihal Engin Vrana(3), Harald Klüter(1), Julia Khzyshkowska(1,2,4)

Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, Germany (1)

- Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, Russia (2) - Protip medical, Strasbourg, France (3) - German Red Cross Blood Service Baden-Württemberg – Hessen, Mannheim, Germany (4)

Patients with Diabetes Mellitus (DM) have elevated blood sugar levels and can suffer from consequences triggered by compromised immune responses. Amongst those consequences are the complications produced by metal implants. As a result of compromised biocompatibility properties, such as metal corrosion triggered by hyperglycemic environment, implant materials generate metal alloy debris, which are the implant wear off particles. The small alloy particles are gradually and constantly released from an implanted material throughout its entire life course. These microparticles and nanoparticles produce negative immune responses and inflammatory complications, which also may be different from initial acute inflammatory phase and rather correspond to chronic inflammation. Here we investigated the effects of hyperglycemic conditions on the inflammatory responses of primary human macrophages exposed to increasing concentrations TiO2 nanoparticles (NPs). We have assessed YKL-40(CHIT3L1) and CCL18(PARK) that correspond to chronic inflammatory factors and are produced in human primary macrophages in response to IFNgamma and IL-4, correspondingly. Furthermore, macrophage ROS levels have been assessed after their stimulation with TiO2 nanoparticle in normoglycemic and hyperglycemic environments.





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P59NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT) ACTS AS A METABOLIC GATE OF SUPPRESSOR MYELOID CELLS MOBILIZATION Cristina Travelli(1) - Sabina Sangaletti(2) - Ubaldina Galli(3) - Mario Paolo Colombo(2) - Armando A. Genazzani(3) - Maria Francesca Consonni(4) - Antonio Sica(5)

Department of Pharmaceutical Sciences, University of Eastern Piedmont, Novara, Italy(1) - Fondazione Irccs Istituto Nazionale Dei Tumori, Fondazione Irccs Istituto Nazionale Dei Tumori, Milano, Italy(2) - University of Eastern Piedmont, 1department of Pharmaceutical Sciences, Novara, Italy(3) - Humanitas Clinical and Research Center, Department of Inflammation and Immunology, Rozzao, Italy(4) - Humanitas Clinical and Research Center, 3department of Inflammation and Immunology, Rozzano, Italy(5)

Cancer induces alteration of hematopoiesis to fuel disease progression. Indeed, perturbation of myelopoiesis in cancer leads to the expansion of immunosuppressive immature myeloid cells, called myeloid-derived suppressor cells (MDSCs). Along with tumor-associated macrophages (TAM), these cells are negative regulators of anticancer immune responses and are considered major promoters of tumor immune evasion, angiogenesis and metastasis formation. Given that reprogramming of energy and amino acid metabolism (e.g. tryptophan and arginine) is an essential event in tumor-related immunosuppression an for MDSCs activation, the aim of our work was to elucidate the role of a crucial enzyme in NAD metabolism called nicotinamide phosphoribosyltransferase (NAMPT) in the function and fate of MDSCs. NAMPT expression increases in various diseases, including chronic inflammatory conditions (e.g. rheumatoid arthritis), metabolic alterations (e.g. diabetes) and cancer. Recently, NAMPT inhibitors have entered clinical trials for solid and non-solid tumors, due to their ability in lowering NAD and ATP levels and, in turn, interfering with malignant cell growth (20). We report that in cancer bearers the macrophage colony-stimulating factor (M-CSF) elevates the myeloid cell levels of NAMPT which acts as negative regulator of the CXCR4 retention axis of hematopoietic cells in the bone marrow. NAMPT inhibits CXCR4 through a NAD/Sirtuin 1-mediated inactivation of HIF-1α-driven CXCR4 gene transcription, unleashing mobilization of MDCSs and enhancing their production of suppressive nitric oxide. Pharmacological inhibition or myeloid-specific ablation of NAMPT prevented MDSCs mobilization, reactivated specific antitumor immunity and enhanced the antitumor activity of immune checkpoint inhibitors. Our finding identifies NAMPT as a metabolic gate of MDSCs mobilization and as new targetable level of intervention to improve cancer immunotherapy.





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P60RESOLUTION OF CUTANEOUS LEISHMANIASIS AND PERSISTENCE OF LEISHMANIA MAJOR IN THE ABSENCE OF ARGINASE 1 Katrin Paduch(1) - Andrea Debus(1) - Ulrike Schleicher(1,2) - Christian Bogdan(1,2)

Institute of Clinical Microbiology, Immunology and Hygiene, Universitätsklinikum Erlangen(1) - Medical Immunology Campus Erlangen, Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Erlangen, Germany(2)

Cutaneous infections of wildtype (WT) C57BL/6 mice with the intracellular protozoon Leishmania (L.) major heal on their own. However, low numbers of parasites will survive lifelong within various host cells. Persisting L. major are contained by inducible NO synthase (NOS2) that metabolizes L-arginine to citrulline and leishmanicidal NO. In latent cutaneous leishmaniasis parasites are found in fibroblasts, which exhibit a reduced ability to express NOS2 and inefficiently kill Leishmania, as well as in NOS2-negative myeloid cells. Both in vitro and in vivo we have observed that expression and activity of NOS2 protein can be antagonized by arginase (Arg)1 which converts L-arginine into urea and ornithine. We therefore hypothesized that persistence of L. major in NOS2 -negative host cells is facilitated by or even requires the expression and activity of Arg1.During acute cutaneous leishmaniasis Arg1 mRNA and protein were clearly present in the skin lesions and draining lymph nodes of L. major-infected WT mice, whereas Arg1 protein became undetectable during the chronic phase of infection. Absence of Arg1 in hematopoietic and endothelial cells (using Tie2Cre+/-Arg1fl/fl C57BL/6 mice) did not alter lesion development or parasite burden in skin and draining lymph nodes during the acute or persistent phase of infection. Also, deletion of Arg1 neither increased the production of NO by NOS2 in situ nor caused a change in tropism for distinct host cell types as assessed by confocal laser scanning fluorescence microscopy. Similar to WT controls, parasites persisting in Arg1-deficient mice favored NOS2-negative areas and mainly resided in fibroblasts or myeloid cells.We conclude that Arg1 expression by hematopoietic and endothelial cells is completely dispensable for the wound healing in cutaneous leishmaniasis and for the long-term survival of L. major which has fundamental implications for our understanding of parasite persistence in vivo.

This study has been supported by the DFG (SFB1181) and the EU/BMBF (Infect-ERA IV).

3 · INFECTIONS




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P61HIF-PROLYLYDROXYLASE-2 (PHD2) IS A FUNDAMENTAL REGULATOR OF NEUTROPHIL MIGRATION IN INNATE IMMUNITY

Sundary Sormendi(1) - Ana Meneses(1) - Iannis Kourtzelis(1) - Pablo Saez(2) - Pablo Vargas(2) - Triantafyllos Chavakis(1) - Ben Wielockx(1)

Institute of Clinical Chemistry and Laboratory Medicine, Technische Universität Dresden (Germany)(1) - Systems Biology of Cell Polarity and Cell Division, Institut Curie (France) (2)

The adaptive response to hypoxia is tightly regulated by Hypoxia Inducible Factor (HIF) prolyl hydroxylase domain proteins (PHD1-3). These oxygen-sensors directly regulate the HIF-activity and orchestrate numerous physiological as well as pathological processes. Moreover, a considerable amount of studies have shown that HIFs can directly control the innate and adaptive immune response in vivo. Although PHD2 has been reported to be the main regulator of the hypoxia pathway, its important role during the immune response is only recently becoming clear. In this study, we utilized different conditional knock-out mice for PHD2 in order to elucidate its effect during local inflammation. Using different well-known local inflammatory models (e.g. inflammatory arthritis, skin irritation and lung inflammation), we were able to demonstrate that loss of PHD2 in the hematopoietic compartment (Vav:cre-PHD2f/f mice) leads to enhanced neutrophil recruitment to the site of inflammation. Using a double knock-out strategy, we revealed this response to be reliant on the overexpression of the downstream effector HIF2α. Further characterization of PHD2-deficient neutrophils showed enhanced cell motility and accumulation of cytoskeletal actin at the front of the cell under mechanical confinement conditions. Accordingly, RNAseq Gene Enrichment Set Analysis reveal positive signatures of cytoskeleton components the implicated in the maintenance of the leading edge and cell migration persistence. Together, these results identify PHD2 as a prominent regulator of HIF-2α-driven neutrophil motility.





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P62THERAPEUTIC OPTIONS FROM THE UNDERSTANDING OF MACROPHAGE HETEROGENEITY IN THE TUMOR MICROENVIRONMENTB. Savino(1,2), E. Setten(1,2), I. Mattiola(1), D. Mavilio(2), M. Locati(1,2)

Humanitas Clinical and Research Center, Rozzano, MI Italy(1) - Department of Medical Biotechnologies and Translational Medicine, Università degli Studi di Milano, Segrate, MI Italy (1)

Innate immunity is naturally endowed with anti-tumoral activities and with protective immune responses against infections. However, the effect of microenvironmental signals and infection-related abnormalities hamper its in vivo potential. Tumor-associated macrophages (TAM) acquire a protumoral M2-like functional profile, which promotes tumor invasion and metastasis and suppresses local immunity by effector cells, such as cytotoxic T lymphocytes and NK cells. Consistent with this, clinical studies have found a correlation between the high macrophage content in tumors and poor prognosis. However, TAM are composed by a number of different subpopulations with different features depending on the variety of factors present in the microenvironment. Indeed, an aberrant macrophage-NK cells crosstalk might favor viral or bacterial infections, as reported in AIDS patients. In particular, the preferential M2 macrophage polarization and the expansion of the dysfunctional CD56neg NK cell subset characterize active and viremic stages of HIV-1 infection. We have reported that a complex network allows human classically activated (M1) macrophages, but not resting (M0) or alternatively activated (M2) macrophages, to prime resting autologous NK cells increasing their cytotoxicity. Furthermore, the M1-induced NK cell production of IFNγ plays a role in the context of rescuing M2 toward a proinflammatory M1 profile to boost innate immune responses against infections and tumor. We are characterizing macrophage heterogeneity in cancer patients and investigating its predictive potential for immunotherapies efficacy. Indeed, a better understanding of macrophage plasticity and in particular of mechanisms underlying NK cell-macrophage crosstalk will pave the way to innovative therapeutic approaches aiming at TAM depletion or their reprogramming in antitumor actors.

2 · CANCER




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P63PHENOTYPIC AND TRANSCRIPTIONAL PROFILING REVEALS MONOCYTE-RELATED MACROPHAGES PHENOTYPICALLY RESEMBLING DC IN HUMAN INTESTINE

L. Richter(1) - O. J. B. Landsverk(1) - N. Atlasy(1) - A. Bujko(1) - S. Yaqub(3) - R. Horneland(4) - O. Øyen(4) - E. M. Aandahl(4) - K. E. A. Lundin(5) - H. G. Stunnenberg(2) - E. S. Bækkevold(1) - F. L. Jahnsen(1)

Department of Pathology and Centre for Immune Regulation, Oslo University Hospital and University of Oslo, Oslo, Norway(1) - Department of Molecular Biology, Faculties of Science and Medicine, Radboud Institute of Molecular Life Sciences, Radboud University, Nijmegen, Netherlands(2) - Department of Gastrointestinal Surgery, Oslo University Hospital, Oslo, Norway(3) - Department of Transplantation Medicine, Section for Transplant Surgery, Oslo University Hospital, Oslo, Norway(4) - Department of Gastroenterology, Oslo University Hospital, Oslo, Norway(5)

The tissue dendritic cell (DC) compartment is heterogeneous, and the ontogeny and functional specialization of human tissue conventional DC (cDC) subsets and their relationship with monocytes is unresolved. Here we identify monocyte-related CSF1R+Flt3- antigen presenting cells (APCs) that constitute about half of the cells classically defined as SIRPα+ DCs in the steady-state human small intestine. These CSF1R+Flt3- APCs express calprotectin and very low levels of CD14, are transcriptionally related to monocytes-derived cells, and accumulate during inflammation. CSF1R+Flt3- APCs show typical macrophage characteristics by being functionally distinct from their Flt3+ cDC counterparts: under steady-state conditions they excel at antigen uptake, show lower migratory potential, and are inefficient activators of naïve T cells. These results have important implications for the understanding of the ontogenetic and functional heterogeneity within human tissue DCs and their relation to the monocyte lineage.





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AUTHORS’ INDEXAE. M. Aandahl ................................... 84Zeinab Abdullah .............................. 57Elisa Albini ........................................ 73Morgan Alexander ........................... 35Andrea Alimonti .............................. 13Meshal Alobaid ................................ 35Martino Ambrosini.......................... 26Petronela Ancuta .............................. 16Cristina Anselmi .............................. 49Francesca Antonini .......................... 76Myriam Aouadi ................................ 19Ryanne Arisz ..................................... 51Anna C. Aschenbrenner ................. 69N. Atlasy ............................................ 84

BChristina Backes ............................... 27E. S. Bækkevold ................................ 84Prachi Bagadia .................................. 72Anna-Lorena Bär.............................. 57Jarek Baran ........................................ 71Jaroslaw Baran .................................. 65Giulia Barbiera ................................. 18Emelie Barreby ................................. 19M. Letizia Barreca ............................ 11Jose Basilio ........................................ 25Emiliano Basini ................................ 11Claudio Bassi .................................... 49Kevin Baßler ..................................... 17Christina Baumgartinger ................ 25Andreas Beilhack ............................. 52Maria Laura Belladonna ................. 70Michele Bellucci ............................... 73Ronan Bergin .................................... 30Andreas Bergthaler .......................... 42Silvio Bicciato ............................. 14, 34E.M. Bignell ...................................... 62Christoph J. Binder .......................... 25Katrina Binger .................................. 57Giovanni Blandino ........................... 38Bruce Blazar ...................................... 34Camille Bleriot ................................. 19Anna Maria Blom ............................. 33Stephan Blueml................................. 63Przemyslaw Blyszczuk ..................... 65Christian Bogdan ............................. 81Maria Carla Bosco ............................ 76Guillaume Bouyer ............................ 51Aline Bozec ....................................... 64Natascha Brigo .................................. 50Carlos G. Briseño ............................. 72Christoph Brochhausen .................. 57Vincenzo Bronte .................. 14, 34, 49Sheila Brown ............................... 28, 41Laura Rosa Brunet ........................... 41Julia Brunner .................................... 25

Julia Stefanie Brunner.... 29, 58 63, 68Davide Brusa ..................................... 37Stefano Bruscoli................................ 27A. Bujko ............................................. 84

CMatilde Cacciatore ........................... 32Arianna Calcinotto .......................... 13Rocco Cappellesso ........................... 46Carmine Carbone ............................ 34Luciano Cascione ............................. 49Amélie Cattin .................................... 16Filippo Cernilogar ........................... 21Tiziana Cestari .................................. 49Nayaret Chamorro ........................... 15Triantafyllos Chavakis ..................... 82Hung-Jen Chen ................................. 22Nadezhda Cherdyntseva ................. 44John Cidlowski ................................. 15Francesco Cilenti .............................. 18Menna R. Clatworthy ...................... 57Mario Paolo Colombo ............... 12, 80Cristina Conforti Andreoni ........... 24Maria Francesca Consonni ............. 80P.C. Cook ........................................... 62Peter Cook ......................................... 28Paul R. Crocker................................. 26James Crooks .................................... 41Stephan Culemann........................... 54D. Cunoosamy .................................. 62Marcin Czepiel ................................. 65

DCharlotte Dahlem............................. 36Jill Dalimot ........................................ 51Marta D’Andolfi................................ 61Hannes Datler ...................... 25, 29, 68Claire Davies ..................................... 59Johann De Bono ............................... 13Andrea Debus ................................... 81Angelo Paolo Dei Tos ...................... 32Marjorie De la Fuente...................... 15Paola Del Bianco .............................. 61Alessandro Della Puppa .................. 61Genny Del Zotto .............................. 76Francesca De Marchis ..................... 73Anna Dembek................................... 43Joke M.M. Den Haan ....................... 26Francesco De Sanctis .......... 34, 37, 49Menno De Winther.......................... 22Balazs Dezso ..................................... 66David Diaz-Jiménez ........................ 15Stefanie Dichtl .................................. 50Britta Diesel ................................ 27, 36Helmut Dolznig ................................ 42Sara Donzelli ..................................... 38Karen Dubois .................................... 15

Nelita Du Plessis............................... 52Vivek Durai ....................................... 72Andrzej Dzionek .............................. 24

EIna Eckert .......................................... 52Matthew Edwards ............................ 28Stéphane Egée ................................... 51Janine Erler ....................................... 47Alessandra Eva .................................. 76

FFrancesca Fallarino .................... 11, 72Matteo Fassan ................................... 34Ambrogio Fassina ............................ 46Francesco Fazi .................................. 38M. Feeney .......................................... 62John R. Ferdinand ............................ 57Alessandra Fiore .................. 34, 37, 49Andrew Flatley ................................. 44Natalia Fonseca Do Rosario ........... 16Giulia Fontemaggi ........................... 38Cecilia Forss ...................................... 28I. Förster ............................................ 62Giulio Fracasso ................................. 34H.A. Franks ....................................... 39Stephanie Deborah Fritsch .. 20, 40, 42Wataru Fujii ...................................... 17

GAlicia Galdon .................................... 41Ubaldina Galli .................................. 80Enzo Gallo ......................................... 38Adrianna Galuszka .......................... 71Alessandra Gamberucci .................. 76Natalio Garbi .................................... 53Stefano Garetto ................................. 37Marco Gargaro ........................... 11, 72Jerzy Gasowski .................................. 65Gil Gasparoni ................................... 43Dominique Gauchat ........................ 16Judy Geissler ..................................... 51Ioanna Gemünd ............................... 69Armando A. Genazzani .................. 80Marco Genua .................................... 18Roman G. Gerlach ........................... 57Amir Ghaemmaghami .................... 35Nikolas Giannakis ............................ 66Mathew Gibbson .............................. 35Florent Ginhoux ............................... 19Rosalba Giugno ................................ 49Annie Gosselin ................................. 16Michele Gottardi .............................. 32Jean-Philippe Goulet ....................... 16Joanna Grabowska ........................... 26Friedrich Grässer.............................. 27Fabio Grassi ...................................... 73




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Guillermo Griffith ............................ 22Federica Grillo .................................. 76Ursula Grohmann ....34, 70, 73, 75, 78Anna Gruca ................................. 65, 71Alexandru Gudima .......................... 79Zofia Gula .......................................... 65

HNina Hachenthal .............................. 27Günter J. Hämmerling ..................... 53Madlen Hansen ................................ 55Anna-Lena Hardt ............................. 69Martin Harmsen ............................... 74David Haschka ................................. 56Silvia Hayer ....................................... 63Akos Heinemann ....................... 31, 77Markus Hengstschläger ............. 20, 40Matthias Hentze ............................... 56Merima Herac ................................... 42Marcela A. Hermoso ........................ 15S.J. Hill ............................................... 39Keli Hippen ....................................... 34Martin Hirtl ...................................... 42Francesca Hofer ................................ 49Alexander Hoffmann ....................... 56Melanie Hofmann ......... 25, 29, 63, 68Laszlo Holasz .................................... 66Leoni Hoogterp ................................ 26Jessica Hoppstädter............. 27, 36, 43Stefanie Hörer ...................... 20, 42, 60R. Horneland .................................... 84Edward Horton ................................. 47Emma Houlder ................................. 28Christian Huck ................................. 56Hanno Huwer ............................. 27, 43Chang-Il Hwang ............................... 47

IAlberta Iacono .................................. 73Manuela Iezzi .................................... 34Dario Iodice ...................................... 18

JA.M. Jackson ..................................... 39F. L. Jahnsen ...................................... 84Katharina Jandl ................................. 31Jonathan Jantsch ......................... 57, 64

KSujin Kang ......................................... 23Theodoros Kapellos ......................... 17Karl Katholnig .................................. 42A.P. Kaur ............................................ 39Martina Kerndl .................... 25, 29, 68Sonja M. Kessler ............................... 36Julia Khzyshkowska ......................... 79Alexandra K. Kiemer .......... 27, 36, 43Barbara Killy ..................................... 55Christian Kirschneck ....................... 64Kento Kitada ..................................... 57Thomas Klei ...................................... 51Harald Klüter ....................... 44, 74, 79

Christoph Koch ................................ 54Karine Köhrer ................................... 60Mariusz Korkosz .............................. 65Iannis Kourtzelis .............................. 82Thomas Krausgruber ....................... 60Elisabeth Kremmer .......................... 44Thomas Kreutzberg ......................... 53Branislav Krljanac ............................ 54Gerhard Krönke ............................... 54Anne Krug ......................................... 45Atsushi Kumanogoh ........................ 23Christian Kurts ................................. 57Mario Kuttke ..................................... 58Julia Kzhyshkowska ................... 44, 74

LAlessia Lamolinara .......................... 34O. J. B. Landsverk ............................. 84Michaela Lang ............................. 40, 42Roland Lang ...................................... 55Irina Larionova ................................. 44Rita Lawlor ........................................ 34Rita Teresa Lawlor ............................ 49Petra Leidinger ................................. 27Marzena Lenart ................................ 65Alexander Lercher............................ 58Clarice Lim ........................................ 60Monika Linke .............................. 20, 42Nikolai Litviakov .............................. 44Tengfei Liu ......................................... 44M. Locati ........................................... 83Giovanna Lollo ................................. 61Maria Lopatniuk .............................. 36K. E. A. Lundin ................................. 84Eleonora Lusito ................................ 18Manfred B. Lutz ................................ 52Andriy Luzhetskyy ........................... 36

MAntonio Macchiarulo ................ 75, 78Andrew Macdonald ................... 28, 41A.S. Macdonald ................................ 62Christoph Magnes ............................ 40Mark A. Magnuson .......................... 42Sara Magri ......................................... 61Giorgia Maltese ................................ 47Susanna Mandruzzato ........ 34, 49, 61Giuseppe Manfroni .......................... 11Giorgia Manni ............................ 11, 72Paola Marcolongo ............................ 76Myriam Martin ................................. 33Jonathan O. Martinez ...................... 48Matteo Martini ................................. 37Silvia Masciarelli .............................. 38Elena Masetto ................................... 61Luca Mastracci .................................. 76Julia Maria Matschinger ............ 42, 60I. Mattiola .......................................... 83D. Mavilio .......................................... 83Emilia Mazza .................................... 14Marta Mazzocco ............................... 37James Mcconnell .............................. 47

Eckart Meese ..................................... 27Davide Melisi .................................... 34Ana Meneses ..................................... 82Christoph Metzendorf ..................... 56Rebecca Metzger .............................. 45Mario Mikula .................................... 42Elisa Milano ...................................... 38Mihaela F. Militaru .......................... 63Marie Minet ...................................... 27Veronique Miron .............................. 59Kondaiah Moganti ........................... 74Roberto Molinaro ............................ 48Giada Mondanelli ...................... 34, 70Elisa Montaldo ................................. 18Frank Ralph Morrissey-Wettey ...... 24Marije Mossel .................................... 74Paul N. Moynagh.............................. 30Martina Muckenthaler .................... 56Dominik N. Müller .......................... 57Kenneth M. Murphy ........................ 72Theresa L. Murphy ........................... 72Peter Murray ..................................... 34T. Musarrat ........................................ 39Laszlo Musiejovsky .......................... 25

NGergely Nagy ..................................... 66Laszlo Nagy ....................................... 66Manfred Nairz .................................. 56Dario Nardi ....................................... 72Roland Naumann ............................. 54Patrick Neubert .......................... 57, 64Wolfgang Neuhofer .......................... 64Lorenzo Nicolè ................................. 46Sebastian Nielsen ............................. 47

OGeorg Obermayer ............................ 25Marie Oestreich ................................ 69Katarzyna Olesek.............................. 26Ewa Oleszycka .................................. 30Mauricio Olivares-Morales ............ 15Ciriana Orabona .............................. 70Daniela Oster .................................... 43Renato Ostuni ................................... 18O. Øyen .............................................. 84

PAgostino Paccagnella....................... 32Katrin Paduch ................................... 81Salvatore Paiella ................................ 49Maria Teresa Pallotta ....................... 73Eleonora Panfili ................... 70, 75, 78Nikolaos Papaioannou .................... 21Daniela Parada ................................. 15Anna Pasto ........................................ 48P.M. Patel ........................................... 39Andreas Patsalos .............................. 66Julian Peltier ...................................... 55Christa Pfeifhofer-Obermair ......... 50A.T. Phythian Adams ...................... 62Laura Pinton ..................................... 61




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Geny Piro........................................... 34Anna Livia Pissavino ....................... 76Carmen Pizarro ................................ 17Ornella Poffe ..................................... 49Andrea Pompa .................................. 73Peter Proff.......................................... 64Magdalena Pruszko.......................... 38Paolo Puccetti ................................... 72Antonia Puchner .............................. 63Andrea Puglisi .................................. 76

QRodrigo Quera .................................. 15

RFederica Raggi .................................. 76Stephan Rambichler ........................ 21Carmen Reitinger............................. 57Roberta Resaz ................................... 76Vladimir Riabov ............................... 44Eliana Ribechini ............................... 52Carlo Riccardi ................................... 27Sarah-Lane Richards........................ 35L. Richter ........................................... 84Amanda Ridley ........................... 28, 62Sonja Rittchen .................................. 31Uwe Ritter ......................................... 52Carla Rodriguez ............................... 14Kathrin Rohrer ................................. 77David Roula ...................................... 77Jean-Pierre Routy ............................. 16Boris Rusev ....................................... 49Magdalena Rutkowska-Zapala ....... 65Vladimir Ryabov .............................. 79

SAndrea Sacconi ................................. 38Pablo Saez .......................................... 82Victoria Saferding ............................ 63Ugur Sahin ........................................ 34Natallia Salei ..................................... 21Johanna Salvermoser ....................... 21Luisa Sambado ................................. 32Maria Sambataro .............................. 32Tiziana Sanavia ................................. 46Sara Sandri .................................. 34, 37Sabina Sangaletti ........................ 12, 80Laura Santambrogio .................. 75, 78Sara Sartori........................................ 49Silvia Sartoris ....................... 34, 37, 49B. Savino ............................................ 83Giulia Scalisi ............................... 11, 72Aldo Scarpa ................................. 34, 49Alessandra Scarponi .................. 75, 78Gernot Schabbauer ....................25, 29, .....................................42, 55, 58, 63, 68Valentin Schatz ................................. 57Barbara Scheiber-Mojdehkar ......... 20Ulrike Schleicher ........................ 52, 81Christine Schmitt-Mbamunyo ....... 53Maria Schöller .................................. 20David Schörghofer ........................... 42

Gunnar Schotta ................................ 21Barbara U. Schraml .......................... 21Agnes Schröder .......................... 57, 64Andrea Schroll .................................. 56Ronja Schuchert ............................... 21Rufina Schuligoi ............................... 31Joachim L. Schultze ......................... 69Joachim Schultze .............................. 17Christian Schulz ............................... 21Marcel H. Schulz .............................. 43Birgit Schütz ................................ 40, 42Lea Seep ............................................. 69Daniela Segalerba ............................. 76Markus Seifert .................................. 50Paolo Serafini .................................... 14E. Setten ............................................. 83Tatjana Sevastyanova ....................... 79Omar Sharif ................... 25, 29, 58, 68Michael Sherratt ............................... 47Antonio Sica ...................................... 80Maciej Siedlar ............................. 65, 71Martin Simon.................................... 43Drik Skowasch .................................. 17Karolina Izabela Smolag .................. 33Josef S . Smolen................................. 63Samantha Solito ................... 34, 49, 61Sundary Sormendi ........................... 82Richard Sparla .................................. 56Clarissa Spataro ................................ 13Andreas Spittler ................................ 42Richard Stange .................................. 63Malgorzata Stec ........................... 65, 71Piero Maria Stefani .......................... 32Gert Storm ........................................ 26Martin Stradner................................ 77H. G. Stunnenberg ........................... 84Eva Sturm .......................................... 77Nyamdelger Sukhbaatar ..... 20, 40, 42André Sulen ...................................... 19Manuela Sushnitha .......................... 48Freya Svedberg ................................. 28F.R. Svedberg .................................... 62

THeribert Talasz ................................. 56Ennio Tasciotti ................................. 48Derek J. Theisen ................................ 72Igor Theurl ........................................ 20Christoph Thiele ............................... 69Melanie Timmen .............................. 63Jens Titze ........................................... 64Stefan Tomiuk ................................... 57Cristina Travelli ................................ 80Claudio Tripodo ............................... 12Rosalinda Trovato ............... 34, 37, 49Piotr Tymoszuk ................................ 50Petros Tzerpos .................................. 66

UStefano Ugel ......................... 34, 37, 49Thomas Ulas...................................... 69Daniela Unterleuthner .................... 42

VLaura Valori ...................................... 32Robin Van Bruggen ......................... 51Dieke Van Dinther ........................... 26Yvette Van Kooyk ............................. 26Antoon Van Oosterhout ................. 28Dimitri Van Simaeys........................ 14Ger Van Zandbergen ....................... 57Luigi Varesio ..................................... 76Pablo Vargas ...................................... 82Fulvia Vascotto ................................. 34Robert Vassallo ................................. 67Francisco Verdeguer ........................ 19Marina Vettore.................................. 61Roberto Vidal ................................... 15David Voehringer............................. 54Andrea Vogel ................. 25, 29, 58, 68Claudia Volpi ....................... 70, 75, 78Daniela Vorholt ................................ 24Nihal Engin Vrana ........................... 79Maja Vujic-Spasic ............................. 56

WVanessa Sue Wacleche ..................... 16Gerhard Walzl ................................... 52Donald T. Weed ................................ 14Kazimierz Weglarczyk ..................... 65Thomas Weichhart ........ 20, 40, 42, 60Andrea Weichselbaum .................... 57Günter Weiss ........................ 20, 50, 56Jakob Weiszmann ............................. 40Ben Wielockx .................................... 82Anna Williams.................................. 59Mark Wilson ..................................... 28Nathalie Wirth .................................. 43Stefan Wirtz ...................................... 54Caroline Wrangler ........................... 53

XXiaojiang Xu...................................... 15

YS. Yaqub ............................................. 84

ZElena Zagato ..................................... 13Lucia Zanatta .................................... 32Marina Zavyalova ............................ 44Serena Zilio ....................................... 14Alessia Zoso ...................................... 14Alicja Zylicz ...................................... 38




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