From the Department of Infectious Diseases and Microbiology of the University of Lübeck Director: Prof. Dr. med. Jan Rupp Progesterone-induced changes to the murine vaginal microbiota and host immunity enhance susceptibility to chlamydial infection Dissertation for Fulfillment of Requirements for the Doctoral Degree of the University of Lübeck from the Department of Natural Sciences Submitted by Nathalie Loeper from Berlin Lübeck 2018 First Referee: Prof. Dr. med. Jan Rupp Second Referee: Prof. Dr. rer. nat. Hauke Busch Date of oral examination: 07.02.2019 Approved for printing: 21.02.2019 Table of content Table of content List of figures .................................................................................................................. V List of tables................................................................................................................... VI Abbreviations ................................................................................................................ VII Abstract ............................................................................................................................ 1 Zusammenfassung .......................................................................................................... 2 1. Introduction ............................................................................................................... 4 1.1 Sexually transmitted infections with Chlamydia trachomatis ........................................ 4 1.1.1 The pathogen Chlamydia trachomatis........................................................... 4 1.1.2 Clinical aspects: treatment and sequels ........................................................ 5 1.1.3 The impact of vaginal microbiota on female health and disease ................... 6 1.2 Experimental design of chlamydial infections ............................................................ 10 1.2.1 Development and characterization of a mouse infection model .................. 10 1.2.2 Host immune response towards chlamydial infections ................................ 12 1.3 The impact of female sex hormones on infectious diseases ...................................... 16 1.3.1 The estrous cycle of rodents ....................................................................... 16 1.3.2 Sex hormones modulate various infections ................................................. 19 2. Aim of the study ...................................................................................................... 21 3. Material and methods ............................................................................................. 22 3.1 Material ..................................................................................................................... 22 3.1.1 Devices ....................................................................................................... 22 3.1.2 Consumables.............................................................................................. 24 3.1.3 Chemicals ................................................................................................... 25 3.1.4 Medical products ........................................................................................ 26 3.1.5 Buffers and solutions .................................................................................. 27 3.1.6 Cultivation media ........................................................................................ 27 3.1.7 Antibodies ................................................................................................... 28 3.1.8 Enzymes ..................................................................................................... 28 3.1.9 Kits ............................................................................................................. 29 I Table of content 3.1.10 Primer for PCR ........................................................................................... 29 3.1.11 Bacterial strains .......................................................................................... 29 3.1.12 Cell lines ..................................................................................................... 30 3.1.13 Mice ............................................................................................................ 30 3.2 Cell culture ................................................................................................................ 30 3.3 Infection biology ........................................................................................................ 31 3.3.1 Cell infection with Chlamydia muridarum .................................................... 31 3.3.2 Recovery assay .......................................................................................... 31 3.3.3 Quantification of Chlamydia ........................................................................ 31 3.3.4 Infection modulation with antibiotics ............................................................ 32 3.4 Microscopy ................................................................................................................ 32 3.4.1 Indirect immunofluorescence of Chlamydia ................................................ 32 3.4.2 Direct immunofluorescence of Chlamydia ................................................... 32 3.5 Animal experiments .................................................................................................. 33 3.5.1 Experimental procedures ............................................................................ 33 3.5.2 Pathology score .......................................................................................... 34 3.6 Microbiota sequencing .............................................................................................. 35 3.6.1 Library preparation...................................................................................... 35 3.6.2 Next-generation sequencing ....................................................................... 37 3.7 Flow cytometry .......................................................................................................... 37 3.7.1 Tissue digestion and preparation of a single cell suspension ...................... 37 3.7.2 Staining ...................................................................................................... 38 3.7.3 Measurements and gating strategy ............................................................. 38 3.8 Histological examination ........................................................................................... 39 3.9 Bioinformatics and statistics ...................................................................................... 40 3.9.1 Software ..................................................................................................... 40 3.9.2 Processing of raw NGS data ....................................................................... 40 3.9.3 Graphical plotting ........................................................................................ 41 3.9.4 Statistical analysis ...................................................................................... 41 4. Results ..................................................................................................................... 42 II Table of content 4.1 Characterization of murine vaginal microbiota and tissue composition ...................... 42 4.1.1 Commensal vaginal microbiota ................................................................... 42 4.1.2 Vaginal tissue composition ......................................................................... 43 4.2 Modulation of the vaginal milieu by sex hormones .................................................... 44 4.2.1 Reduced immune cells in uterine tissue after progesterone and β-estradiol treatment .................................................................................................... 44 4.2.2 Vaginal microbiota was altered in mice treated with progesterone, but not with β-estradiol ................................................................................................... 45 4.2.3 Gut microbiota was unaffected by progesterone treatment ......................... 48 4.3 Progesterone enhanced susceptibility of mice to genital C. muridarum infection ....... 49 4.3.1 Enhanced bacterial shedding by progesterone-treatment ........................... 49 4.3.2 Progesterone-treated mice developed pathology ........................................ 50 4.3.3 Vaginal microbiota during chlamydial infection ............................................ 52 4.3.4 Infection-induced host immune response was elevated in progesterone-treated mice .......................................................................... 54 4.4 Application of antibiotics – effects on Chlamydiae and microbiota ............................. 56 4.4.1 Antibiotic efficacy in vitro ............................................................................ 56 4.4.2 Efficacies of antibiotics varied in eradicating chlamydial infection ............... 57 4.4.3 Ampicillin affected vaginal microbiota but not chlamydial infection .............. 58 5. Discussion ............................................................................................................... 62 5.1 Sex hormones alter site specific microbiota .............................................................. 62 5.1.1 Commensal vaginal microbiota shape a stable vaginal milieu ....................