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Lysophosphatidic Acid-Mediated GPR35 Signaling in CX3CR1+ Macrophages Regulates the Intestinal Cytokine Milieu

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Lysophosphatidic Acid-Mediated GPR35 Signaling in CX3CR1+ Macrophages Regulates the Intestinal Cytokine Milieu Lysophosphatidic acid-mediated GPR35 signaling in CX3CR1+ macrophages regulates the intestinal cytokine milieu Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Berna Kaya aus der Türkei 2020 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Christoph Hess Prof. Dr. Jan Hendrik Niess PD Dr. Cristian Riedel Basel, 23.06.2020 Prof. Dr. Martin Spiess The Dean of Faculty of Science Table of Contents TABLE OF CONTENTS ABBREVIATIONS .................................................................................................................. IV ABSTRACT ........................................................................................................................... VII 1 INTRODUCTION ................................................................................................................. 1 1.1 The intestinal immune system ................................................................................... 1 1.1.1 Innate immunity in the intestine ............................................................................ 1 1.1.1.1 Intestinal epithelium....................................................................................... 2 1.1.1.2 Mononuclear phagocytes ............................................................................... 5 1.1.2 Host- and microbiota-derived metabolites ........................................................... 10 1.2 Inflammatory bowel diseases .................................................................................. 14 1.2.1 Host immune system and genetics ...................................................................... 15 1.2.2 Microbiome ........................................................................................................ 15 1.2.3 Diet and other environmental factors .................................................................. 16 1.2.4 Current therapy options ...................................................................................... 17 1.3 G protein-coupled receptor (GPR) 35 .................................................................... 19 1.4 Aims of the study ..................................................................................................... 22 2 METHODS ........................................................................................................................ 23 2.1 Human samples ....................................................................................................... 23 2.2 Animals .................................................................................................................... 25 2.2.1 Mouse lines ........................................................................................................ 25 2.2.2 Generation of Gpr35-IRES-tdTomato knock-in mice .......................................... 25 2.2.3 Construction of Gpr35-flox and -knockout (KO) mice ........................................ 27 2.2.4 Zebrafish lines .................................................................................................... 29 2.3 In vivo experiments .................................................................................................. 31 2.3.1 Dextran sodium sulfate-induced colitis mouse model .......................................... 31 2.3.2 Mouse endoscopy ............................................................................................... 31 2.3.3 Treatment of zebrafish with 2,4,6-Trinitrobenzenesulfonic acid .......................... 31 2.3.4 Treatment of zebrafish with antibiotics ............................................................... 32 2.3.5 Treatment of mice with antibiotics ...................................................................... 32 2.3.6 Challenging of mice with E. coli-CFP ................................................................. 32 2.3.7 Exposure of zebrafish with Vibrio anguillarum ................................................... 32 2.3.9 LPA treatment of zebrafish larvae for cytokine expression analysis .................... 33 2.3.8 In vivo macrophage migration assay in zebrafish ................................................ 33 2.4 Histology and imaging ............................................................................................. 33 2.4.1 Preparation of RNA probes for Gpr35b in zebrafish ........................................... 33 2.4.2 In situ hybridization for gpr35b in zebrafish ....................................................... 34 2.4.3 GPR35 staining on human biopsies and mouse tissues ........................................ 35 2.4.4 Hematoxylin-eosin (H&E) staining and histological scoring ............................... 36 2.4.5 Autotaxin staining of mouse colon ...................................................................... 36 2.4.6 Ex vivo imaging of mouse tissues ........................................................................ 36 2.5 Cell isolation ............................................................................................................ 37 III Table of Contents 2.6 Antibodies, cell staining, and flow cytometry ......................................................... 37 2.7 RNA extraction and quantitative PCR ................................................................... 40 2.8 RNA sequencing ...................................................................................................... 41 2.9 In vitro assays ........................................................................................................... 42 2.9.1 3’-5’-Cyclic adenosine monophosphate (cAMP) assay ....................................... 42 2.9.2 Mouse bone marrow-derived macrophages (BMDMs) ........................................ 43 2.9.3 Trans-well migration assay for mouse BMDMs .................................................. 43 2.9.4 Enzyme-linked immunosorbent assay (ELISA) for corticosterone ...................... 44 2.9.5 LPA ELISA using ex vivo colonic explants of DSS-treated mice ........................ 44 2.10 Western blot LPA- or LPS- treated BMDMs ....................................................... 44 2.11 Identification of GPR35T108M and TNF blocker response in IBD patients .......... 45 2.12 Statistical analysis.................................................................................................. 45 3 RESULTS ........................................................................................................................... 46 3.1 Colonic macrophages express GPR35 .................................................................... 46 3.1.1 Gpr35b is predominant in the intestine of zebrafish ............................................ 46 3.1.2 Gpr35 is expressed in the human and mouse gastrointestinal tract ...................... 48 3.1.3 Macrophages in the intestinal lamina propria are GPR35+ ................................... 48 3.1.4 GPR35+ and GPR35- colonic macrophages are transcriptionally distinct ............. 51 3.2 Regulation of GPR35 by the microbiota and intestinal inflammation .................. 52 3.2.1 Gpr35 expression is microbiota-dependent in zebrafish and mice ....................... 52 3.2.2 Intestinal inflammation elevates GPR35 expression in zebrafish and mice .......... 54 3.2.3 Bacterial infection or colonization induces GPR35 in the intestine ...................... 55 3.2.4 Increased number of GPR35+ cells in patients with ulcerative colitis .................. 56 3.3 Lysophosphatidic acid signaling depends on GPR35 in macrophages .................. 57 3.3.1 LPA inhibits the cAMP release in humanGPR35-transfected cells ...................... 57 3.3.2 LPA leads to GPR35-dependent Tnf induction in zebrafish ................................. 58 3.3.3 GPR35-deficiency leads to altered LPA signaling in BMDMs ............................ 59 3.3.4 LPA facilitates GPR35-dependent chemotaxis of macrophages........................... 63 3.4 Intestinal inflammation enhances Autotaxin and LPA production ....................... 64 3.5 Macrophage-expressed GPR35 is protective in DSS colitis mouse model ............. 67 3.5.1 GPR35-deficient mice have augmented DSS-induced colitis ............................... 67 3.5.2 Macrophage-specific GPR35 deletion leads to aggravated colitis ........................ 68 3.5.3 LPA attenuates DSS colitis in a macrophage-expressed GPR35 manner ............. 70 3.5.4 Gpr35ΔCx3cr1 mice have enhanced neutrophil infiltration during DSS colitis ........ 71 3.5.5 GPR35 deletion in macrophages causes reduced TNF production in colitis ......... 73 3.6 TNF attenuates colitis and induces corticosterone synthesis in Gpr35ΔCx3cr1 mice 74 3.6.1 TNF is protective in Gpr35ΔCx3cr1 mice during DSS colitis .................................. 74 3.6.2 TNF induces Cyp11b1 and corticosterone synthesis in Gpr35ΔCx3cr1 mice ........... 76 3.6.3 TNF does not influence DSS colitis severity in WT mice .................................... 77 3.7 GPR35T108M variant might correlate with TNF blocker responses in IBD ............ 78 4 DISCUSSION ..................................................................................................................... 79 4.1 Heterogeneous expression of GPR35 in intestinal lamina propria monocytes and macrophages .................................................................................................................
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