Relevance of decidual dendritic cells in peri-implantation period as biosensors of altered endometrial stromal secretome.

S GORI , E SOCZEWSKI, A SCHAFIR, E GRASSO, L FERNÁNDEZ , A GRASSI BASSINO, J BLEJER, J PRESA , F SARAVIA, D VOTA, G MARTÍNEZ, C PÉREZ LEIRÓS AND R RAMHORST.

Houston

Congreso; American Society of Reproductive Immunology; 2023

American Society of Reprodcutive Immunology

Relevance of decidual dendritic cells in peri-implantation period as biosensors of altered endometrial stromal secretomeSoledad Gori 1, Elizabeth Soczewski 1, Ana Schafir 1, Esteban Grasso 1, Laura Fernández 1, Alejandra Grassi Bassino 2, Jorgelina Blejer 2, Jessica Presa 3, Flavia Saravia 3, Daiana Vota 1, Gustavo Martínez 4, Claudia Pérez Leirós 1 & Rosanna Ramhorst 1.1Laboratorio de Inmunofarmacología, IQUIBICEN-CONICET, Departamento de Química Biológica, FCEN-UBA, Buenos Aires, Argentina, 2Fundación Hemocentro de Buenos Aires, Argentina, 3 CONICET, Instituto de Biología y Medicina Experimental (IByME). Buenos Aires, Argentina, 4 Fertilis Medicina Reproductiva, Buenos Aires, ArgentinaProblem: During decidualization, endometrial stromal cells undergo physiological endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) allowing a sterile inflammatory response to achieve a successful implantation. However, recent evidence indicates that an imbalance of the stromal ERS/UPR is associated with reproductive failures. In this sense, stromal cells exposed to soluble factors from low-quality embryos suffer exacerbated ERS/UPR. Recently, we showed that decidualization induces DC-10 differentiation, a novel subset of immunoregulatory dendritic cells (DCs). Interestingly, DCs can sense stress signals from other cells which condition their immune functional profile. Here we focused on the impact of the altered secretome from stressed stromal cells on tolerogenic DC conditioning. Method: Human Endometrial Stromal Cell (HESC) line was treated or not with a potent ERS-inducer thapsigargin (Tg) for 4h or decidualized by medroxiprogesterone and dibutyryl cAMP for 8 days (Dec). In parallel, Dec cells were also stimulated with pooled embryo-conditioned media (ECM) obtained from normal or impaired developing embryos (ND-Dec or ID-Dec, respectively). In all cases, HESC were cultured for 48h to collect conditioned media (CM). Then, CM were added during monocyte differentiation to dendritic cells (rhGM-CSF+rhIL-4, 5 days). Expression of tolerogenic markers was evaluated by flow cytometry and ELISA and ERS/UPR pathways by RT-qPCR. Results: On one hand, the tolerogenic DC-10 profile observed in DC cultures differentiated with Dec-CM in the presence of ECM was sustained, with higher frequency of CD14+CD86lowHLA-G+ cells. However, the embryo quality affected the secretory profile of DC-10: DC-10 cultures conditioned with ID-Dec displayed lower IL-10 secretion compared to ND-Dec and higher TNF-α production after LPS stimulation. These results suggest that in the presence of an arrested embryo, DCs could differentially shape the immunological microenvironment contributing to its clearance during the menstrual phase. On the other hand, we confirmed that DCs can sense ERS signals from endometrial stromal cells. Soluble factors released by stressed stromal cells (Tg-CM) inhibited the CD1a+CD14- immature DC differentiation and induced a pro-inflammatory profile on DC cultures, increasing CD86hi cell-population, COX2 expression and IL-1β secretion. Moreover, the transmission of ERS and the differential modulation of three UPR pathways in DCs conditioned by Tg-CM were observed. The expression of IRE1α, PERK and ATF4 increased, whereas ATF6α diminished. Even the splicing of the UPR marker XBP1 was increased in Tg-CM though at low levels and without changes in its nuclear translocation in comparison to Non-Tg-CM cultures, suggesting the triggering of terminal UPR despite of adaptive UPR. In line with this, we observed increased percentage of AnnexinV+/PI- and AnnexinV+/PI+ cells in Tg-CM cultures, suggesting a lytic programmed cell death. Finally, we tested the negative impact of stressed-DC profile on blastocyst implantation. Using a blastocyst-like spheroids migration model, we confirmed an altered trophoblast migration in the presence of supernatants from DCs differentiated with Tg-CM in comparison with non-stressed CM. Conclusions: Stromal cells can transmit reticular stress signals to DCs inducing alterations in their functional profile that could impact negatively on embryo implantation.