Spatial immune dynamics in mucosal HIV infection

<p dir="ltr">Sexual transmission is the most common mode of HIV infection, making the female genital and rectal tracts critical mucosal sites for preventing HIV transmission. Various factors, such as local inflammation and disruption of the epithelial barrier, have been suggested to increase the risk of sexual HIV transmission. Despite the significance of the female genital tract in this context, it remains less studied compared to other key sites like the gastrointestinal tract. Additionally, the long-term impact of HIV on the female genital tract is still not fully understood. Therefore, this thesis aims to investigate factors influencing HIV susceptibility and the long-term effects of chronic HIV infection on the female genital tract, utilizing a multi-omics approach that integrates conventional in situ bioimaging, spatial proteomics, and transcriptomics.</p><p dir="ltr">Topical application of microbicides presents a promising strategy for preventing HIV in at-risk individuals. However, the failure of earlier microbicides underscores the need for more thorough safety evaluations. In <b>Paper I</b>, we performed an exploratory safety assessment following the topical rectal application of the promising microbicide Q-Griffithsin. We developed in situ bioimaging analysis workflows to evaluate its impact on the sensitive rectal epithelium and the CD4+ HIV target cell population. No adverse effects were observed on the rectal epithelium or on the frequency, density, and spatial distribution of CD4+ cells, indicating that Q-Griffithsin is safe based on these criteria.</p><p dir="ltr">Depot medroxyprogesterone acetate (DMPA), a widely used hormonal contraceptive in HIV-endemic regions, has been linked to an increased risk of HIV acquisition, possibly due to induced inflammation, epithelial disruption and an altered genital microbiome, though the evidence remains conflicting. In <b>Paper II</b>, we used a multi-omics approach to thoroughly investigate the effects of DMPA on the cervical mucosa and its potential impact on HIV susceptibility. No differences in the cervical microbiome were detected at either the clinical or molecular level. Our results did however demonstrate a significant epithelial disruption, and an enhanced immunological profile associated with long-term DMPA use, both of which are factors linked to increased HIV susceptibility.</p><p dir="ltr">Chronic systemic immune activation is a hallmark of HIV infection, but its effects on the female genital tract remain poorly understood. In <b>Paper III</b>, we performed a comprehensive analysis of the cervical transcriptional landscape in the context of chronic HIV infection and its impact on the cervical epithelium. Our transcriptional analysis revealed significant immune activation associated with HIV infection, primarily driven by interferon signaling and T cell activity. Additionally, we observed notable epithelial disruption, which was further validated through in situ bioimaging. These findings suggest a potential mechanistic link between cervical immune activation and epithelial instability, with important implications for HIV-related comorbidities in the female genital tract.</p><p dir="ltr">In <b>Paper IV</b>, we extended our investigation of the T cell-related immune activation and epithelial disruption observed in previous studies, using the cutting-edge techniques, spatial transcriptomics and multi-epitope ligand cartography. Spatial transcriptomics revealed a tissue-wide induction of antibody-related genes, suggesting the recruitment of B cells or plasma cells into the cervical mucosa in association with HIV infection. Consistent with the findings from <b>Paper III</b>, we also observed significant induction of interferonrelated genes, alongside more sporadic activation of T cell-related genes. In situ bioimaging showed a marked expansion of tissue-resident memory CD8+ T cells and recruitment of peripheral CD8+ T cells. Interestingly, no changes were observed in the CD4+ T cell population, indicating that the systemic and gastrointestinal depletion of CD4+ T cells in HIV-infected individuals is not mirrored in the cervix of HIV-infected women.</p><p dir="ltr">In conclusion, this thesis has explored factors associated with HIV infection and susceptibility in the rectal and female genital tracts using a multi-omics approach. We developed bioimage analysis workflows to assess epithelial integrity in both the single-layered rectal mucosa and the multi-layered stratified epithelium of the ectocervical mucosa. The findings provide new insights into immune activation, immune cell localization, and epithelial disruption linked to HIV susceptibility and infection. Notably, this is the first study to apply both bulk RNA sequencing and spatial transcriptomics to the female genital tract of HIV-infected women. Together, these results have important implications for HIV infection and prevention, potentially contributing to the development of more effective prevention strategies and targeted interventions for women at risk of, or living with, HIV.</p><h3>List of scientific papers</h3><p dir="ltr">I. A topical rectal douche product containing Q-Griffithsin does not disrupt the epithelial border or alter CD4+ cell distribution in the human rectal mucosa. <b>Franzén Boger M, </b>Benhach N, Hasselrot T, Brand RM, Rohan LC, Wang L, McGowan I, Edick S, Ho K, Meyn L, Matoba N, Palmer KE, Broliden K, Tjernlund A<i>. </i>Scientific Reports. 2023 May 9;13(1):7547. <br><a href="https://doi.org/10.1038/s41598-023-34107-w" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41598-023-34107-w</a></p><p dir="ltr"><br></p><p dir="ltr">II. Multi-omics analysis of the cervical epithelial integrity of women using depot medroxyprogesterone acetate. Bradley F, <b>Franzén Boger M</b><i>,</i> Kaldhusdal V, Åhlberg A, Edfeldt G, Lajoie J, Bergström S, Omollo K, Damdimopoulos A, Czarnewski P, Månberg A, Oyugi J, Kimani J, Nilsson P, Fowke K, Tjernlund A, Broliden <i>K. </i>PLoS Pathogens. 2022 May 9;18(5):e1010494<br><a href="https://doi.org/10.1371/journal.ppat.1010494" rel="noreferrer" target="_blank">https://doi.org/10.1371/journal.ppat.1010494</a></p><p dir="ltr"><br></p><p dir="ltr">III. Sustained immune activation and impaired epithelial barrier integrity in the ectocervix of women with chronic HIV infection. <b>Franzén Boger M</b>, Hasselrot T, Kaldhusdal V, Miranda G, Czarnewski P, Edfeldt G, Bradley F, Rexaj G, Lajoie J, Omollo K, Kimani J, Fowke K, Broliden K, and Tjernlund A.<i> </i>[Submitted]</p><p dir="ltr">IV. Spatial transcriptomics and <i>in situ </i>immune cell profiling of the host ectocervical landscape of HIV infected Kenyan sex working women. <b>Franzén Boger M</b>, Kaldhusdal V, Pascual-Reguant A, Kroh S, Uecker R, D. Burgener A, Lajoie J, Omollo K, Kimani J, Fowke K, E. Hauser A, Tjernlund A, and Broliden K.<i> </i>[Submitted]</p>