Discussion
In the current study, we developed a method to sort live Th17 cells from PF of severe endometriosis patients to evaluate the function of pathological Th17 cells. We are the first to use CXCR3, CCR4, and CCR6 isolate PF Th17 cells from endometriosis patients. In the future, it would be necessary to detect more surface markers to precisely pinpoint Th17 cells. Furthermore, detection of the above chemokine receptors would recognize Th1, Th2, and Th1/Th17 subsets and thus greatly facilitates research on T cell biology at endometriosis lesions.
Th17 cells are heterogeneous and possess a remarkable degree of plasticity [16]. Our study indicates that PF Th17 cells are heterogeneous according to the expression of CD27 and IL-17RE. CD27 is a member of TNF receptor family expressed on CD4+ T cells at the priming or effector stage, while its ligand, CD70, is expressed on activated dendritic cells and lymphocytes [17]. Recent research suggests its inhibitory effect on Th17 cells through repressing transcription of IL-17 and CCR6 [15]. IL-17RE forms a receptor complex with IL-17RA to bind to IL-17C to potentiate the Th17 cell response [14]. IL-17C is preferentially secreted by epithelial cells in the lung, skin, and colon [18]. Interestingly, the Human Protein Atlas describes high IL-17C expression in the endometrium (https://www.proteinatlas.org/ENSG00000124391-IL17C/tissue), strongly suggesting the existence of IL-17C in endometriosis lesions. Our ongoing research is investigating the levels of IL-17C in PF and endometriosis tissues. Besides, our data indicate that IL-17RE rather than CD27 marks a more active Th17 subpopulation, because IL-17RE+ Th17 cells expressed more Th17-related cytokines and were more proliferative than IL-17RE-Th17 cells. A possible explanation is that IL-17RE-triggered signaling overcomes CD27-induced signaling.
Through RNA-Seq, we found that most of the top 20 up-regulated metabolic genes in IL-17RE+ Th17 cells encode enzymes involved in OXPHOS and ETC, while most of the down-regulated metabolic genes were associated with other metabolic pathways. It has been known that mitochondrial respiration is enhanced during Th17 differentiation and OXPHOS is essential for Th17 cell pathogenic signature gene expression [19]. Besides, Th17 cells have limited capacity to promote glycolysis to react to metabolic stresses and they rely on OXPHOS for the production of energy and cytokines [20]. Indeed, we observed higher pro-inflammatory activity associated with higher ATP and ROS in IL-17RE+ Th17 cells. This suggests that OXPHOS and ETC support the pathogenicity of PF IL-17RE+ Th17 cells in endometriosis. Our data also poses an interesting question: whether the IL-17C/IL-17RE axis results in the up-regulation of OXPHOS and ETC-related genes. This could be answered in future studies.
What remains unknown is the origin of the heterogeneous PF Th17 subsets. Are they a CD4+ T cell population at different maturation statuses? Or do they arise from different CD4+ T cell subsets under distinct microenvironment factors? Lineage tracing of Th17 cells in animal models and single-cell transcriptome sequencing might give the answer.
In conclusion, this study provides a novel method to detect and isolate live PF Th17 cells from endometriosis patients and unveils the functional and metabolic heterogeneity of PF Th17 subsets. Furthermore, the data of other differentially expressed genes shed light on the elucidation of other molecular mechanisms that modulate the function of pathological Th17 cells in endometriosis.