Monocytesanddendritic cells: rolesduringhumaninfluenza andhantavirusinfections

The majority of HIV infections today occur through sexual HIV transmission. The female genital mucosa offers a barrier against incoming pathogens. Although, studies show that the vaginal microbiome, co-current infections and local inflammation, the use of hormonal contraceptives and microbicides, can weaken this protective lining. In this thesis in situ digital image analysis workflows were developed and used together with protein profiling, to characterize the effects of such factors on the genital mucosal barrier integrity and the immune cells therein. Topically applied microbicides can protect against HIV. In paper I we introduced image analysis as a refined tool for evaluation of microbicide safety. We confirmed that a promising microbicide candidate, Q-GRFT, had no negative effect on the rectal epithelium while causing a small, but probably biologically negligible, increase in CD4+ HIV target cells. We also discovered potential effects of multiple biopsy sampling that should be considered when designing pre-clinical studies. Specific tissue resident immune cells with an effector memory phenotype have a rapid response against re-infections and may be important against HIV infection. In paper II, we observed that HIV infected women had increased levels of CD103-CD8+ tissue resident memory cells compared to uninfected women, and that this may be due to a recent influx of these effector cells that have not yet upregulated the CD103 retention molecule. In paper III we revealed that women taking the hormonal contraceptive DMPA, had a thinner superficial layer of the female genital mucosa. Lack of this protective layer in combination with having more HIV target cells located closer to the vaginal lumen, could contribute to the increased HIV risk in women taking DMPA. In paper IV we discovered that Lactobacillus non-iners dominated women had a more intact epithelium, and Gardnerella dominated women had a different spatial localization of CD4+ cells in the epithelium. Secreted protein profiles from Lactobacillus dominant women had elevated levels of anti-inflammatory and epithelial barrier proteins compared to non-Lactobacillus dominant women. These factors may contribute to reduced HIV risk in Lactobacillus-dominant women. The results of this thesis highlight the benefits of using digital image analysis as a tool for studying spatial and structural changes in the mucosal tissue barrier and the immune cell landscape therein. We showed potential mechanisms in how different factors increase the HIV risk. These findings will support development of interventions aimed to strengthen the mucosal barrier, and thereby reduce transmission of sexually transmitted infections. LIST OF SCIENTIFIC PAPERS I. Impact of Q-Griffithsin anti-HIV microbicide gel in non-human primates: In situ analyses of epithelial and immune cell markers in rectal mucosa. Gökçe Günaydın*, Gabriella Edfeldt*, David A. Garber, Muhammad Asghar, Laura Noȅl-Romas, Adam Burgener, Carolina Wählby, Lin Wang, Lisa C. Rohan, Patricia Guenthner, James Mitchell, Nobuyuki Matoba, Janet M. McNicholl, Kenneth E. Palmer, Annelie Tjernlund and Kristina Broliden. Scientific Reports. 2019 Dec 2; 9:18120. *These authors contributed equally. II. HIV-infected women have high numbers of CD103-CD8+ T cells residing close to the basal membrane of the ectocervical epithelium. Anna Gibbs, Marcus Buggert, Gabriella Edfeldt, Petter Ranefall, Andrea Introini, Stanley Cheuk, Elisa Martini, Liv Eidsmo, Terry B. Ball, Joshua Kimani, Rupert Kaul, Annika C. Karlsson, Carolina Wählby, Kristina Broliden and Annelie Tjernlund. The Journal of Infectious Diseases. 2018 Aug 1; 218:453-65. III. Digital tissue image analysis reveals apical distribution of HIV target cells in the ectocervical epithelium of women using depot medroxyprogesterone acetate. Gabriella Edfeldt, Julie Lajoie, Maria Röhl, Kenneth Omollo, Mathias Mack, Joshua Kimani, Julius Oyugi, Carolina Wählby, Keith R. Fowke, Kristina Broliden and Annelie Tjernlund. Manuscript. IV. Cervicovaginal microbiota affects the human ectocervical epithelial barrier structure as determined by in situ digital image analysis and protein profiling. Gabriella Edfeldt, Frideborg Bradley, Sofia Bergström, Julie Lajoie, Jiawu Xu, Vilde Kaldhusdal, Alexandra Åhlberg, Behnaz K.H. Binizy, Cecilia Mattsson, Anna Månberg, Carolina Wählby, Joshua Kimani, Julius Oyugi, Peter Nilsson, Keith R. Fowke, Annelie Tjernlund, Douglas S. Kwon and Kristina Broliden. Manuscript. Publication not included in the thesis Increased Cervical CD4+CCR5+ T Cells Among Kenyan Sex Working Women Using Depot Medroxyprogesterone Acetate. Julie Lajoie, Annelie Tjernlund, Kenneth Omollo, Gabriella Edfeldt, Maria Röhl, Geneviève Boily-Larouche, Julianna Cheruiyot, Makubo Kimani, Joshua Kimani, Julius Oyugi, Kristina Broliden and Keith R. Fowke. AIDS Research and Human Retroviruses. 2019 Mar 5; 10.1089. CONTENTS 1 Aim of thesis . 11 2 The current HIV epidemic . 12 2.1 In numbers . 12 2.2 High risk groups . 12 2.3 HIV transmission . 13 3 Epithelial barrier – the wall of protection . 14 3.1 The unique female genital mucosa . 14 3.2 Morphology of the squamos stratitifed epithelium . 15 3.3 Epithelial junction proteins . 15 3.4 Immune cells of the ectocervix . 16 3.4.1 CD4+ T cells . 17 3.4.2 Langerhans cells . 17 3.4.3 Tissue resident cells . 17 4 The vaginal microbiome . 19 4.1 Bacterial vaginosis . 19 4.1. 1 Bacterial vaginosis and inflammation . 19 4.1. 2 Bacterial vaginosis risk factors . 20 4.2 Lactobacilli . 20 4.2.1 Lactobacillus crispatus vs. Lactobacillus iners . 21 4.3 Gardnerella . 21 4.4 Prevotella . 22 4.5 The gut microbiome . 22 5 Hormonal influences on the female mucosa . 23 6 HIV prevention and microbicides . 24 6.1 HIV prevention . 24 6.2 Microbicides . 24 6.3 Microbicides and mucosal damage . 24 6.4 Microbicide safety testing . 25 6.5 Prevent clinical study . 25 6.5. 1 Lead compound Griffithsin, an HIV-binding lectin . 25 6.5. 2 Pre-clinical testing . 26 6.5. 3 In situ analysis . 26 7 Digital tissue image analysis . 27 7.1 The process . 27 7.2 Human vs. computer . 28 7.3 Importance of studying tissue . 28 8 Methodological considerations . 29 8.1 Sample material and ethical aspects . 29 8.2 Immunostaining . 30 8.3 Image analysis . 30 8.3.1 Software . 31 8.3.2 Image preprocessing and segmentation . 31 8.3.3 Compartmentalization. 34 8.3.4 Feature extraction and selection . 35 8.4 Microbiome analysis . 35 8.4.1 Nucleic acid extraction . 35 8.4.2 16S variable region .