ABSTRACT: We aimed to gain a comprehensive understanding of transcriptional profiles of circulating Th1, Th1/17, and Th17 cells in endometriosis patients (n=10) as compared to healthy controls (n=12). Briefly, fresh peripheral blood was collected from subjects, peripheral blood mononuclear cells (PBMCs) were isolated using density centrifugation and then cryopreserved until later bulk analysis. PBMCs were later thawed, CD4+ T cells were magnetically isolated (#17952, StemCell Technologies, Canada) and stimulated with 30ng/mL of PMA and 1ug/mL of Ionomycin and incubated for 4hrs. Cells were then stained for FACS and sorted into viable populations of Th17 (CD3+CD4+IFNg-IL-17+), Th1/17 (CD3+CD4+IFNg+IL-17+), and Th1 (CD3+CD4+IFNg+IL-17-) cells using BD FACSAria™ III. RNA was then isolated from Th1, Th1/17, and Th17 cell populations from patients and controls using a commercially available kit (#74004, Qiagen, Canada). Samples were then quality checked and sent for bulk RNA sequencing (RNAseq) at McGill Genome Centre (MGC, Canada) using Illumina NovaSeq X Plus 10B (Illumina, USA) and subsequent bioinformatic analyses, as per standard pipelines. Briefly, analyses included principal component analysis (PCA), unsupervised and supervised clustering, heatmap generation, differential expression analysis (DEG), volcano plots, fold-change distribution, and Gene Ontology (GO) enrichment analyses. RNA sequencing revealed extensive Th subset reprogramming in endometriosis, most predominately in Th17 cells (2,220 DEGs). Collectively, our findings reveal significant immune remodeling in endometriosis and highlight a distinct, aberrant Th17 cell phenotype, contributing to endometriosis pathophysiology. Ultimately, this work supports the repurposing of IL-23- and IL-17-targeted therapeutics to provide novel options for endometriosis patients, as these therapeutics have been successfully used in other chronic inflammatory diseases and may also target endometriosis-associated comorbidities.
