Serum Response Factor is Essential for Endometrial Function and Prevention of Inflammatory Fibrosis Based on Single Cell Analysis

Ryan M. Marquardt¹, Sara A. Grimm², Peter F. Lais¹, San-Pin Wu¹, Eunhee M. Jeong³, Bruce A. Lessey⁴, Jae-Wook Jeong³, John P. Lydon⁵, Francesco J. DeMayo¹

1.   Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

2.   Biostatistics and Computational Biology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

3.   Department of Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, MO, USA

4. Department of Obstetrics and Gynecology, Wake Forest Baptist Health, Winston-Salem, NC, USA

5. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA

Abstract Text:

Healthy pregnancy requires a dynamic and supportive uterine environment facilitated by the differentiation of endometrial stromal fibroblasts into decidual cells and tight regulation of inflammation. Inflammatory uterine disorders like endometriosis and endometritis are associated with both infertility and fibrotic tissue stiffness, but the cell type-specific mechanisms that generate endometrial inflammation and fibrosis remain unclear. Mesenchymal action of the widely expressed transcription factor serum response factor (SRF) plays a known role in lung fibrosis, and analysis of publicly available data revealed that endometrial SRF gene expression was reduced in patients with recurrent implantation failure. We therefore hypothesized that SRF regulates the transcriptional environment needed for endometrial homeostasis and early pregnancy success. To test this hypothesis, we analyzed primary patient samples, assayed human endometrial stromal cells in vitro, and performed single cell RNA-seq analysis of conditional uterine Srf knockout mice. Immunohistochemical analysis showed that SRF protein expression was decreased in endometrial biopsies from infertile patients with endometriosis compared to healthy controls. RNAi-based SRF knockdown in human endometrial stromal cells resulted in a blunted response to decidualizing hormone treatment. Transcriptomic analysis of these cells showed that SRF deficiency disrupted cytoskeletal remodeling and growth response genes, and validation experiments confirmed decreases in F-actin levels and in cell viability. Upon conditional ablation of Srf in the mouse uterus with the progesterone receptor (Pgr)-cre model, Pgr^cre/+Srf^f/f (Srf^d/d) females were infertile, displaying uterine fibrosis and failure to decidualize or support embryo implantation. Single cell RNA-seq analysis of the preimplantation stage pregnant uterus demonstrated dramatic changes in endometrial cell populations of Srf^d/d mice compared to controls. Uterine Srf ablation caused massive myeloid immune cell infiltration (29-fold increase of monocytes, 21-fold increase of neutrophils, 3-fold increase of dendritic cells, and 2-fold increase of macrophages). Subclustering followed by differential expression analysis demonstrated that Srf^d/d stromal fibroblasts downregulated the decidualization-related genes Egr1 (-54-fold), Fst (-10-fold), Scara5 (-9-fold), and Zbtb16 (-5-fold). Srf^d/d basal fibroblasts downregulated cytoskeletal genes Acta2 (-15-fold), Tagln (-8-fold), and Cnn2 (-7-fold). Proliferating fibroblast populations shifted from 64% G2-M phase in controls to 81% S-G2 phase in Srf^d/d uteri. These changes demonstrate a disruption of pre-decidual growth response and cytoskeletal integrity in Srf-deficient fibroblasts similar to the human stromal cell findings but do not provide an obvious explanation for the inflammatory fibrosis phenotype. To our surprise, Srf^d/d luminal epithelial cells overexpressed estrogen-responsive innate inflammatory genes including Sprr2b (332-fold), Cxcl5 (172-fold), Lcn2 (150-fold), C3 (49-fold), Cxcl2 (41-fold), and Ltf (34-fold), unveiling the probable driver of myeloid immune infiltration. Similarly, Srf^d/d glandular epithelial cells upregulated Lcn2 (153-fold) and Cxcl5 (39-fold) while showing diminished expression of gland marker Spink1 (-137-fold). Mmp7, a gene with a known neutrophil-attractant, pro-fibrotic role in lungs increased 280-fold in luminal epithelium and 26-fold in glandular epithelium. Immunostaining confirmed the presence of F4/80+ macrophage and Ly6G+ neutrophil accumulation localized to epithelial layers in the Srf^d/d uterus. These results demonstrate that SRF is critical for endometrial homeostasis and function in support of pregnancy establishment. While SRF plays a part regulating endometrial stromal fibroblast growth response and cytoskeletal remodeling, its unexpectedly prominent role in vivo is to suppress epithelium-linked innate inflammation. We are now actively investigating these inflammatory paracrine signaling interactions prompted by Srf ablation to determine their mechanistic linkages to steroid hormone signaling and endometrial fibrosis development.