miRNA/mRNA analysis of Chlamydia trachomatis-infected endometrial biopsies indicate increased TGF-β pathways drive epithelial-mesenchymal transition and regulatory T cell differentiation

Chlamydia trachomatis genital tract infection is linked to severe reproductive complications in women, including ectopic pregnancy, infertility, and adverse pregnancy outcomes. Mouse models of infection suggest that chlamydia-induced dysregulation of microRNAs (miRNAs) can drive harmful cytokine responses, pathogenic epithelial-mesenchymal transition (EMT), and fibrosis. To investigate these mechanisms in humans, we profiled miRNA and mRNA expression in endometrial biopsies from women with endometrial infection (Endo+) and compared them to profiles from women with cervix-only infection (Endo-) or no infection. Ingenuity Pathway Analysis (IPA) revealed that Endo+ tissues had upregulated genes associated with innate and adaptive immune response pathways, as well as EMT regulation, while downregulated genes were linked to cell cycle control. An integrative miRNA-mRNA analysis, which combined a review of published miRNA regulation in human infections and immune responses with IPA’s miRNA target filter, identified differentially expressed miRNAs that modulate these pathways in the endometrium of Endo+ women. Functional annotation of these miRNAs showed a predominance of downregulated miRNAs that typically suppress EMT and regulatory T cell (Treg) differentiation, along with miRNAs that usually enhance Th17 responses. Comparisons with previously identified mRNA pathways in blood samples from women with endometrial Chlamydia infection indicated that alterations in TGF-β signaling and EMT were specific to the endometrium. Overall, the miRNA-mRNA interactions inferred from Endo+ tissue suggest increased activity in TGF-β pathways that promote enhanced EMT and Treg differentiation, while reducing Th17 activation. These changes highlight a dual potential for promoting tissue scarring while dampening inflammatory responses that could otherwise limit infection. Endometrial biopsy samples were obtained from cisgender female participants enrolled in two independent cohorts with high exposure to Ct infection. Total RNA was extracted from endometrial biopsies stored at -80°C in tubes containing RNA/DNA Shield (Zymo Research, Irvine, CA). After thawing, samples were weighed, minced, and processed for simultaneous DNA and RNA extraction using the Quick DNA/RNA™ isolation kit according to the manufacturer's protocol (Zymo) with on column DNase I treatment prior to total RNA elution. Libraries were prepared using the Ovation SoLo RNA-Seq kit (NuGen Technologies) and sequenced on an Illumina HiSeq2500 platform in the High Throughput Sequencing Facility (HTSF) at the University of North Carolina.