Single-cell transcriptomics of cervical epithelial responses to HPV-Chlamydia infections and gastroesophageal junction evolution

The cervix of the female reproductive tract (FRT) plays a critical role as a barrier against pathogen invasion, maintaining the sterility of the upper reproductive tract. However, continuous exposure to external stressors such as acidic environments and microbes increases the risk of infections and mucosal stress or injury. The outermost region of the cervix, known as the ectocervix, is particularly susceptible to high microbial loads compared to the inner endocervix. The transition zone (TZ), where the ectocervix's multi-layered stratified squamous epithelium meets the endocervix's single-layered columnar epithelium, is highly prone to infections and cancer development. Human papillomavirus (HPV) is a primary cause of cervical cancer, specifically targeting the stratified epithelium of the ectocervix. Additionally, cofactors such as co-infection with other pathogens and immune suppression play critical roles in the progression of HPV-induced cancers. However, the mechanisms underlying the cohabitation of these pathogens and their role in neoplastic progression remain unclear. While investigating the host-pathogen interaction is crucial, it's equally important to acknowledge the diverse composition of epithelial cell types in the cervix, each with unique roles and gene profiles. Hence, a comprehensive understanding of the innate immune profiles of these cells is essential to understand the perturbations caused by infections and evaluate pathogen susceptibility across ecto- and endocervical regions. Furthermore, the gastroesophageal junction is analogous to the TZ in the cervix, where the stratified squamous epithelium of the esophagus meets the glandular columnar epithelium of the stomach. Much like the cervix, this area is highly susceptible to cancers, often preceded by metaplasia. Chronic gastric reflux leads to esophageal mucosal damage, triggering a unique tissue adaptation where the native squamous epithelium of the esophagus is replaced by columnar epithelia, resulting in Barrett’s esophagus (BE), increasing the risk of esophageal cancers with poor prognosis. Despite its critical importance in the pathophysiology of gastrointestinal diseases, understanding the molecular mechanisms of epithelial homeostasis in this region and the role of the underlying stroma in maintaining it could significantly enhance our knowledge of gastrointestinal tract pathogenesis. Additionally, this understanding could provide valuable insights into the similarities and differences between the cervix and gastroesophageal transition zones. In the first part of this thesis, I investigated the systemic effects of HPV and Chlamydia trachomatis coinfection on host epithelia using patient-derived 3D cervical organoid models that mimic in vivo tissue architecture. To mimic persistent HPV infection, HPV16 E6E7 oncogenes were integrated into the genome of ectocervical epithelial stem cells and generated mature organoids. These HPV16 E6E7 organoids were coinfected with C. trachomatis and subjected to bulk transcriptomic analysis. Each of these pathogens induces similar and unique alterations to host signaling, contributing to a mutagenic microenvironment. Interestingly, I observed an increased stemness and reduced differentiation programs induced by both pathogens in the infected epithelia, highlighting their potential to reprogram host cells and induce dysplastic changes. Further, through comparative analysis with clinical cervical intraepithelial neoplasia (CIN) transcriptome, I found a strong correlation between the transcriptional signatures of HPV E6E7 and coinfection with C. trachomatis induced alteration similar to CIN transcriptional signatures. These results indicate that both pathogens HPV and C. trachomatis can drive preneoplastic changes in ectocervical epithelial cells, and their coinfection can contribute to a synergistic effect. Notably, it is found that the susceptibility to infections varies between ecto- and endocervical epithelia, although the reasons behind this are unclear. In the second part of my thesis, I addressed this knowledge gap by conducting single-cell RNA sequencing (scRNA-seq) experiments employing cervical organoid infection models. Through comparative transcriptomic analysis, I deciphered cellular heterogeneity and lineage development within both ecto- and endocervical epithelia and provided insights into the transcriptional factor profiles and intercellular communication dynamics during C. trachomatis infection. I identified basal squamous stem cells, parabasal cells, and terminally differentiated populations within the ectocervix. In contrast, beyond the conventional secretory and ciliated cells in the endocervix, I also identified stem cell compartment, transitional cells, and a population with robust antigen-presenting and anti-microbial secretion profiles. Following C. trachomatis infection, we observed increased susceptibility and disruption of ectocervical organoids over time, while endocervical organoids remained intact. scRNA-seq analysis also revealed such similarity with increased expression of genes associated with tissue integrity and maintenance in both ecto- and endocervix post-infection. But strikingly, I found that a specific subset of the columnar epithelium of the endocervix is transcriptionally remodeled with high IFN response, immunomodulatory signaling, and defensin secretion. Remarkably, we showed that these secreted defensin molecules were found to reduce the infectivity of C. trachomatis. The insights on the similarities and distinctions between the epithelial subpopulations within these two regions and how these different epithelial subtypes respond to pathogens will be a valuable resource for designing therapeutics to effectively target or prime specific cell types against infections, thereby advancing clinical interventions. In the final part of my thesis, I investigated the evolution dynamics of gastroesophageal squamocolumnar junction (GE-SCJ) using scRNA-seq analysis of mice tissues at different developmental stages, including embryonic day 15 (E15), E19, newborn (pup), and adult stages. I identified the cellular heterogeneity, distinct transcriptional states, and signaling pathways in the esophagus and stomach epithelial and mesenchymal compartments driving development from embryonic to adult stage. My analysis revealed the presence of a unique precursor cell population (Sox11+Vcan+Cldn6+) during embryonic stages that gave rise to p63+Krt5+ squamous epithelia and Krt7+Krt8+ glandular columnar epithelia during development. Strikingly, I found that this population gradually diminished during development and was absent in the adult stage, indicating that adult epithelial stem cells are lineage-committed and are, in turn, responsible for maintaining tissue homeostasis in the GE-SCJ. However, the epithelial tissue architecture, regeneration, repair, and homeostasis are primarily supported by the underlying fibroblast subsets. Given the lack of information on tissue-resident fibroblasts and their spatial organization and role in specifying epithelial lineages during GE-SCJ histogenesis and homeostasis, I defined stromal and epithelial cellular subpopulations and conducted a comprehensive profiling of various morphogenetic signaling pathways, including WNT, BMP, FGF, and PDGF, at different developmental time points. My findings revealed the establishment of distinct WNT signaling microenvironments crucial for proper esophageal growth (WNT-suppressed, as observed in the ectocervix) and stomach growth (WNT-dependent, similar to the endocervix). Additionally, we demonstrated the spatial localization of identified fibroblast subtypes and epithelial subpopulations and validated signaling directionality for some pathways during the pre- and postnatal stages. These insights underscore the significance of niche signaling and offer valuable groundwork for understanding the evolution and maintenance of the SCJ, thereby shedding light on cellular remodeling processes contributing to metaplasia and cancer development.