Vaginal Bacteria Elicit Acute Inflammatory Response in Fallopian Tube Organoids

Abstract

To facilitate in vitro mechanistic studies in pelvic inflammatory disease and subsequent tubal factor infertility, we sought to establish patient tissue derived fallopian tube (FT) organoids and to study their inflammatory response to acute vaginal bacterial infection. FT tissues were obtained from four patients after salpingectomy for benign gynecological diseases. We introduced acute infection in the FT organoid culture system by inoculating the organoid culture media with two common vaginal bacterial species, Lactobacillus crispatus and Fannyhessea vaginae. The inflammatory response elicited in the organoids after acute bacterial infection was analyzed by the expression profile of 249 inflammatory genes. Compared to the negative controls that were not cultured with any bacteria, the organoids cultured with either bacterial species showed multiple differentially expressed inflammatory genes. Marked differences were noted between the Lactobacillus crispatus infected organoids and those infected by Fannyhessea vaginae. Genes from the C-X-C motif chemokine ligand (CXCL) family were highly upregulated in Fannyhessea vaginae infected organoids. Flow cytometry showed that immune cells quickly disappeared during the organoid culture, indicating the inflammatory response observed with bacterial culture was generated by the epithelial cells in the organoids. In summary, we have shown that patient tissue derived FT organoids respond to acute bacterial infection with upregulation of inflammatory genes specific to different vaginal bacterial species. FT organoids is a useful in vitro model system to study the host-pathogen interaction during bacterial infection.

Keywords: Fallopian tube organoids; Host-pathogen interaction; Pelvic inflammatory disease (PID); Tubal factor infertility; Vaginal bacteria.

Figure 1.. Characteristic appearance of Fallopian tube organoids.

A. Morphology remains the same after 4 passages. B-C. H&E staining showing columnar epithelium with cilia (arrows) facing towards the lumen. D-E. IHC with PAX8 (brown) labeling secretory cells, tubulin (blue) labeling cilia. Dashed box in D is shown in higher magnification in E. F. IHC with PAX8 (pink) labeling secretory cells, E-cadherin labeling basal membrane. Scale bars: A: 200 μm; D: 100 μm; others: 20 μm.

Figure 2.. Experimental design and representative samples.

A. Experimental design. B-C. Representative pictures showing morphology of FT organoids remained unchanged before (B) and after (C) 24 hours of culturing with either bacterial species.

Figure 3.. Heatmap overview of gene expression levels of all samples as grouped by treatment groups.

Top panel (grey left bar): negative controls without bacterial co-culture. Middle panel (orange left bar): organoids co-cultured with L. crispatus. Bottle panel (green left bar): organoids co-cultured with F. vaginae. Individuals are color coded in second left bar (organoid ID). Each row is a sample. Each column is a gene. Heatmap scale: −4 to 4 (blue to orange) indicates low to high level of gene expression.

Figure 4.. Differential gene expressions between treatment groups as shown in volcano plots.

A. No significant gene expression changes caused by adding PBS in the culture media. B-C. Bacterial co-culture (B. L. crispatus, C. F. vaginae) resulted in multiple gene expression changes compared to negative controls. D. Differentially expressed genes (in red) between the two bacterial culture groups.

Figure 5.. Disappearance of immune cells in FT organoid culture.

The percentage of viable CD45+ immune cells were analyzed by flow cytometry at various time points during the organoid culture. Gating strategy for each sample; left panel: gating of single cells by forward and side scatter, center panel: gating of live cells (Live/DEAD negative), right panel: gating of CD45+ cells. A. Positive control using PBMCs. B. Day 0 of FT tissue collection, 22% of cells were immune cells. C. Day 1 of 3D organoid culture, 11% of cells were immune cells. D. Day 5 of 3D organoid culture, 1% of immune cells remaining. E. Day 8 of 3D organoid culture, immune cells were undetectable.