| dc.description.abstract | The maternal local immune system is key to a successful pregnancy. Maternal immunity is required to initiate pregnancy related processes, whilst controlling and being active against any infections or malignancies that may occur. For these reasons, imbalanced immune activity in the endometrium can severely impact on pregnancy outcomes.
A master regulator of mucosal immunity is IL-17A, a cytokine produced by immune cells in response to bacterial and fungal infections. This pro-inflammatory cytokine induces epithelial and stromal cells to secrete antimicrobial peptides (AMPs), chemokines and matrix metalloproteases, responsible for infection clearance. IL-17A is also linked to several immune-mediated diseases such as psoriasis, arthritis, multiple sclerosis, where its highly pro-inflammatory signature is responsible for the symptomatology associated with these diseases, making this cytokine an excellent therapeutic target. The possible roles of IL-17A in female reproductive tract are poorly explored. It has been previously shown that women with unexplained infertility who failed to sustain a successful pregnancy after Assisted Reproductive Technologies (ART) showed higher level of IL-17A, both in the endometrium and systemically in blood circulation, therefore we aimed to explore the role of this cytokine in female fertility.
The IL-17 cytokines, IL-17A - IL-17F, share similar structures and functions. We hypothesized that IL-17A and the other cytokines belonging to the same family might have evolved functions related to female fertility in mammalian clades. To investigate this, synteny mapping, Multiple Sequence Alignment (MSA) and phylogeny were applied to the IL-17s belonging to representative species of the three mammalian clades (eutherian, metatherian and prototherian), confirming the similarities in the genomic organisation and in the protein sequences of all mammalian IL-17s. Furthermore, analysis of fertility-related datasets showed an upregulation of IL-17A transcript in metatherian pregnancy stages corresponding to placentation, whereas in eutherian mammals IL-17D expression is increased during placentation. Analyses also demonstrated upregulation of IL-17B and IL-17D in endometriosis, recurrent implantation failure (RIF) and unexplained infertility when compared with healthy women, further emphasising a role for IL17 in female reproductive immunity.
The main producers of IL-17A are classically thought to be lymphoid populations such as TH17, γδT-cells or ILC3 cells. Having shown that IL-17A was increased in our cohort of women with unexplained fertility, immune cell gene signature analysis of bulk RNA-seq from endometrial biopsies revealed that immune cell populations were similar in women with successful and unsuccessful pregnancies. We therefore hypothesised that endometrial epithelial cells were responsible for production of IL-17A in endometrial tissue from women with unexplained infertility. We focused initially on the Ishikawa immortalised endometrial epithelial cell line. Treatment of these cells with bacterial lipopolysaccharide (LPS) or the synthetic analogue of viral double stranded RNA virus poly(I:C) induced upregulation of IL-17A mRNA. IL-12B which in conjunction with IL-23A stimulates activation of RORγt, the transcription factor responsible for IL17 transcription, was also induced and flow cytometry confirmed RORγt protein positive staining in Ishikawa cells. Stimulation with recombinant IL-17A induced increased expression of AMPs and CXCL8 in both Ishikawa cells and primary human endometrial epithelial cells (hEECs), obtained from endometrial biopsies.
Since IL-17A is produced in response to bacterial infections, we explored the uterine microbial composition in women with unexplained infertility from our study cohort. Bacterial DNA was extracted from endometrial biopsies taken from our cohort of women and subjected to 16S sequencing. This analysis identified a more diverse microbiome in the women with unsuccessful pregnancy outcome, with Corynebacterium spp. and Prevotella spp. significantly higher in those women. Given that a greater microbial diversity in bacterial vaginosis results in increased SCFAs, we wondered what effect SCFA might have on endometrial cells. Butyrate treatment of Ishikawa and hEECs cells induced increased expression of AMPs, cytokines such as TNFα and IL-17A, and chemokines, CXCL8. Butyrate treatment led also to the production of IL-17A, IL-8 and TNFα protein levels, confirming the ability of non-immune cells to produce IL-17A. By using selective inhibitors of HIF1α and Nf-κB, we demonstrated that these two pathways seem to be involved in butyrate induction of cytokines, chemokines and AMPs. Analysis of a chromatin immuno-precipitation (ChIP)-seq database identified a butyrylation binding site upstream of the IL-17A gene, which also seemed to be confirmed by performing ChIP on Ishikawa cells treated with butyrate.
A role for IL-17A and butyrate in implantation was explored using models recapitulating the window of implantation. The changes induced during the window of implantation were mimicked by treating cells with progesterone with or without IL-17A. The presence of IL-17A during the maturation of epithelial cells seems to not impact on their receptivity, given that markers of endometrial receptivity such as SPP1 and ITGAV show no changes in expression when IL-17A is added to the progesterone. However, IL-17A slightly reduced expression of stromal decidualisation markers such as PRL or SPP1. Butyrate seems to facilitate stromal cells decidualisation, as can be seen by the increase in PRL and IGFBP1 expression when butyrate is added to the decidualisation media. Also, butyrate was shown to drive endometrial receptivity markers, as it can be noticed a significantly increased Expression of SPP1 and ITGAV is significantly increase. IL-15 and LIF, which are two markers used for detecting the window of implantation are also induced by butyrate in epithelial cells, but are decreased by butyrate in stromal cells.
In summary, we present evidence that IL-17A and other cytokines belonging to the same family, have roles in female fertility and that their expression is dysregulated in fertility complications. Furthermore, we find that endometrial epithelial cells contribute to local mucosal immune activity by producing IL-17A, contributing to a pro-inflammatory environment which is detrimental to fertility. Women who are not able to conceive despite ART had a different endometrial microbiome, which is likely to alter local metabolite environment. Among the stimulants that induce IL-17A production by endometrial epithelial cells in FRT is butyrate, a metabolite derived from microbial species, that can potentially regulate IL-17A expression by epigenetic modification. Also, high butyrate levels during endometrium maturation are associated with increased stromal cell decidualisation and epithelial receptivity markers. The mechanisms regulating the first stages of pregnancy are complex thus, further investigation on the local microbiome-induced immune mechanisms may provide novel therapeutic targets to improve female fertility. | en |
