State of the art
The uterine cervix is a critical tissue barrier between the uterine cavity and vagina. It protects the upper female genital tract from the invading pathogens, while supporting the commensal microbes, thus maintaining the healthy tissue homeostasis. Endocervix is lined by single-layered glandular and mucus-producing columnar epithelium. Ectocervix is lined by stratified squamous epithelium that express distinct cytokeratins and high glycogen in the differentiated cells thus providing nutrition to the commensal microbes and creating an impermeable multilayered defense barrier against invading pathogens [1, 2] (Figure 1). These two epithelial regions meet at sqaumo-columnar junction. Chlamydia and human papillomavirus (HPV) are amongst the highly prevalent pathogens infecting the uterine cervix. These infections often remain asymptomatic , however, they contribute to various pathologies including cervicitis. Further, their coinfections are seen at an increased incidence in cervical cancer patients . Most of the host-chlamydia or HPV interaction studies were demonstrated in genetically unstable cancer cell lines such as HeLa cells derived from cervical adenocarcinoma patient or immortalized cell lines, adapted to invitro cell culturing, which often fail to reflect the true in vivo physiological interactions. Also, how these pathogens interact and establish co-infection in a complex three-dimensional in vivo epithelial tissue is completely unclear. Therefore, patient-derived 3D primary organoid and air-liquid interphase cultures are physiologically relevant models to investigate host-pathogen interactions.
Chlamydia and HPV modulates a range of host cell functions and signaling pathways, including those involved in preserving cellular and genomic integrity [4,5]. To understand the pathogenesis during Chlamydia, HPV and co-infections that represent the near physiological situation, we have established and defined niche factors that facilitate long-term ex vivo propagation of human and mouse squamous stratified ecto- and columnar endocervical epithelial tissue as 3D organoids (Figure 1a-b) as well as 3D air-liquid interface cultures [6,7]. Further, human ectocervical organoids were genetically modified by introducing HPV E6E7 oncogenes into the genome. Using these models, we demonstrate that while HPV E6E7 oncogene expression induce host DNA damage response, Chlamydia infection suppresses the expression of these DNA repair genes. Under these conditions, DNA repair in Chlamydia-infected cells is more prone to errors thus posing the risk of accumulation of mutations . Further, the production of native high-risk HPV virus in quantities that can be used for research is still a difficult and expensive task as it relies on the squamous stratified epithelium for productive life cycle and the release of the native virus into the lumen. We have developed methodologies to use our organoid models to produce the high-risk native HPV virus (Figure 1c) to study native HPV-host interactions.
Figure: a) Schematic of uterine cervix merging at endo-ecto cervix epithelial junction. b) Human ectocervical stem cell derived 3D stratified squamous epithelial organoid with p63 positive basal stem cells that differentiate towards the lumen (left), human endocervix derived Ecaderin (CDH1) positive and p63 negative columnar organoid (right). C) Air liquid interface cultures (left), Organoid (right) derived from ectocervical stem cells produce native high-risk HPV. d-e) scRNA-seq of healthy mouse ectocervix, endocervical epithelium identified two major epithelial types: squamous type (sq) and columnar type (Co). (d) UMAP of epithelial subpopulations color-coded by cluster annotation. (e) Dot plot showing expression of marker genes within epithelial subclusters. Circle size indicates the percentage of cells where the gene was detected. Filled color depicts the normalized and scaled mean expression level.
In this project, we aim to investigate the co-habitation and effects of HPV, Chlamydia and co-infections on the squamous and columnar epithelium of the cervix. Towards this we would first further optimize our method to produce high titers of native high-risk HPV using 3D organoid models for infection analysis. We will investigate how these complex 3D epithelial tissue models facilitate the life cycle of HPV and/or chlamydia, and how infections are established with respect to the type of cells they infect. In particular, stem cells that are exposed on the outer surface of the stratified organoids provide ideal condition to study infections of the stem cell compartment. While ALI cultures are amenable to infect the luminal-differentiated cells.
Integration of HPVE6E7 genes into infected host genome is a key event associated with pathogenesis; however, it is not clear what drive the process. We will investigate if co-infection with Chlamydia contributes to HPV persistence by promoting integration of HPV E6E7 oncogenes into the host cells as well as the role of these oncogenes in Chlamydia persistence or chronic infection. To visualize the persistent form of Chlamydia, we will use the TIMER-chlamydia, an ideal tool that distinguishes the metabolically active and inactive chlamydia. Using advanced super-resolution microscopy, we intend to obtain in-depth visualization of the host-HPV/Chlamydia and HPV-Chlamydia interactions within the whole epithelial organoids in three dimensions. Further, we can visualize the alterations in the cellular and tissue morphology.
Our recent scRNA-seq analysis of ecto and endocervical epithelium revealed cellular subsets within these epithelial types (Figure1 d-e). By performing the single cell transcriptomics, we will investigate how these pathogens influence the cellular composition within the 3D epithelial tissue and how a specific cell type responds transcriptionally to the infection, in particular what changes are induced to the stem and differentiated cells that enable or resist the pathogen colonization. Additionally, we will investigate pathogen transcriptional adaptation to cell type they infect. We would further perform a comparative analysis of chlamydia and HPV infection dynamics of endocervical columnar and ectocervical squamous stratified epithelia.
1. Fadare, O. and A. A. Roma (2019). Normal Anatomy of the Uterine Cervix. Atlas of Uterine Pathology. Cham, Springer International Publishing: 193-196.
2. Tock EP, Shilkin KB. Histochemical study of mucosubstances and glycogen of the postmenopausal human cervix uteri. Am J Obstet Gynecol. 1970;107(2):194–201. doi:10.1016/0002-9378(70)90585-5
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5. Chumduri, C., et al., Subversion of host genome integrity by bacterial pathogens. Nat Rev Mol Cell Biol, 2016. 17(10): p. 659-73.
6. Zadora, P.K., Chumduri et al., Integrated Phosphoproteome and Transcriptome Analysis Reveals Chlamydia-Induced Epithelial-to-Mesenchymal Transition in Host Cells. Cell Rep, 2019. 26(5): p. 1286-1302 e8.
7. Chumduri, C., et al., Transition of Wnt signaling microenvironment delineates the squamo-columnar junction and emergence of squamous metaplasia of the cervix. bioRxiv, 2018: p. 443770. In revision at Nature,
7. Koster.S,et al....Chumduri C, 3D Human cervical organoid model revealed Chlamydia counter HPV oncogenes induced DNA damage response. MS in preparation.