The Impact of Bacterial Signaling Molecules on the Alveolar Immunity of the Lung Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München Raphaël Prungnaud München 2019 Aus dem Comprehensive Pneumology Center (CPC) Institut für Lung Biology and Diseases (iLBD) Impact of Bacterial Signaling Molecules on the Alveolar Immunity of the Lung Diese Dissertation wurde angefertigt unter der Leitung von meines Doktorvaters Prof. Dr. rer. nat. Anton Hartmann am Helmholtz Zentrum München und der Ludwig‐Maximilians‐Universität München. Erstgutachter: Prof. Dr. rer. nat. Anton Hartmann Zweitgutachter: Prof. Dr. Elisabeth Weiß Tag der Abgabe: 14.10.2019 Tag der mündlichen Prüfung: 23.06.2020 ERKLÄRUNG Ich versichere hiermit an Eides statt, dass meine Dissertation selbständig und ohne unerlaubte Hilfsmittel angefertigt worden ist. Die vorliegende Dissertation wurde weder ganz, noch teilweise bei einer anderen Prüfungskommission vorgelegt. Ich habe noch zu keinem früheren Zeitpunkt versucht, eine Dissertation einzureichen oder an einer Doktorprüfung teilzunehmen. München, den 14.10.2019 Raphaël Prungnaud 2 “It’s a marathon, not a sprint.” ‒Everyone ever 3 TABLE OF CONTENTS FIGURES .................................................................................................................... 8 TABLES .................................................................................................................... 10 SUPPLEMENTARY FIGURES ................................................................................ 11 ABSTRACT .............................................................................................................. 12 ZUSAMMENFASSUNG ............................................................................................ 15 INTRODUCTION ..................................................................................................... 19 1. Breathing the world ........................................................................................ 19 1.1. The lung biology and function ................................................................. 19 1.2. The microbes, friends and foes ................................................................. 19 2. The immunology of the lung ........................................................................... 20 2.1. Innate response ........................................................................................ 21 2.2. Adaptive/acquired response ..................................................................... 21 3. The lung epithelial barrier/epithelium ............................................................ 22 4. Macrophages development and origin ............................................................. 24 4.1. Macrophages polarization and functions .................................................. 25 4.1.1. M1: The classical activation .............................................................. 25 4.1.2. M2(a-b-c): The alternative activation ............................................... 26 4.2. Alveolar macrophages: Guardians of the lung homeostasis ...................... 28 5. Th cells: role and differentiation ...................................................................... 28 5.1. Th1/Th2 paradigm ................................................................................... 29 5.2. Th17/Tregs paradigm ................................................................................. 29 6. Lung injury and inflammation ........................................................................ 30 7. The Aryl hydrocarbon Receptor (AhR) and the immune system .................... 32 8. The lung microbiome ...................................................................................... 32 8.1. Pseudomonas aeruginosa and the quorum sensing compounds N-Acyl homoserine lactones ............................................................................................ 33 8.2. The inflammatory response triggered by microbes ................................... 35 4 8.3. Lactobacillus casei and D-tryptophan ...................................................... 36 9. Project outline and hypotheses ....................................................................... 37 MATERIALS AND METHODS ................................................................................ 39 1. Materials ......................................................................................................... 39 1.1. Mice ......................................................................................................... 39 1.2. Kits .......................................................................................................... 39 1.3. Chemicals ................................................................................................. 40 1.4. Recombinant proteins and antibodies ...................................................... 40 1.5. Buffers and solutions ................................................................................ 41 1.6. Primer sequences ...................................................................................... 43 2. Methods .......................................................................................................... 45 2.1. Alveolar macrophages (AM) cell line culture ........................................... 45 2.2. Alveolar epithelial cell type 2 (AECII)..................................................... 46 2.3. Alveolar macrophages polarization ........................................................... 46 2.4. N-Acyl homoserine lactones (AHL) treatment ......................................... 46 2.5. AECII/AM single well coculture .............................................................. 47 2.6. AECII/AM/Pseudomonas aeruginosa (PAO1) transwell culture ............. 47 2.7. AECII/PAO1 supernatant culture wound healing assay .......................... 47 2.8. Intratracheal instillation .......................................................................... 48 2.8.1. Acute Lung Injury (ALI) .................................................................. 48 2.8.2. D-Trp/AHL Treatments ................................................................... 48 2.9. BAL analysis ............................................................................................ 48 2.9.1. Cytospins slides generation ............................................................... 48 2.9.2. May-Grünwald-Giemsa staining ........................................................ 49 2.10. Enzyme-Linked Immunosorbent Assay (ELISA) ...................................... 49 2.11. Cell viability assay with Water Soluble Tetrazolium salt (WST) ............ 49 2.12. RNA isolation .......................................................................................... 50 2.13. Reverse Transcription of total mRNA ..................................................... 50 2.14. Transcriptome analysis – Quantitative PCR ........................................... 50 2.15. Lung homogenate preparation .................................................................. 50 5 2.16. Th cells isolation and differentiation ........................................................ 51 2.17. FACS analysis .......................................................................................... 51 2.17.1. IL-4/17 .............................................................................................. 51 2.17.2. Annexin V-PI .................................................................................... 51 RESULTS: EFFECT OF BACTERIAL METABOLITES ON LUNG EPITHELIUM AND IMMUNITY ..................................................................................................... 52 I- EFFECT OF N-ACYL HOMOSERINE LACTONE COMPOUNDS OF GRAM-NEGATIVE BACTERIA ON LUNG EPITHELIUM AND IMMUNITY ..... 52 1. The quorum sensing molecule AHL modulates Th cell differentiation ............. 52 1.1. 3-oxo-C12-HSL increased IL-17 accumulation in Th17 cells ...................... 52 1.2. 3-oxo-C12-HSL did not influence Th2 cells polarization ............................ 55 2. Bacterial AHL-compounds influence the innate inflammatory response .......... 57 2.1. AHL reduces alveolar macrophages pro-inflammatory gene expression upon M1 polarization .......................................................................................... 57 2.2. AHL signaling increases in vitro inflammatory cytokine production. ....... 58 2.3. In vitro AHL supports M2 polarization ................................................... 60 2.4. Dimethyl sulfoxide influences AM polarization ........................................ 61 3. AHL treatment on AEC/AM coculture reduces M1 inflammatory gene expression ............................................................................................................... 62 4. AHL-molecules impaired epithelial barrier function and wound healing is independent to LPS induced acute inflammatory effect ......................................... 65 5. P. aeruginosa PAO1 supernatant impairs epithelial wound healing independently to 3-oxo-C12-HSL ............................................................................. 69 6. In vivo Acute Lung Injury (ALI) can be modulated by bacterial effectors ...... 71 7. Application of QS-molecules failed to reduce LPS induced neutrophil recruitment ............................................................................................................. 72 II- EFFECT OF D-TRP OF PROBIOTIC GRAM-POSITIVE BACTERIA