The inflammatory response during the implantation period: association with the Reticular Stress and the Unfolded Protein Response

E. GRASSO, S. GORI, E. SOCZEWSKI, L. FERNÁNDEZ, L. GALLINO, T. F. LOBO, S. DAHER, C. M. IRIGOYEN, G. MARTINEZ, C. RUHLMANN, C. PÉREZ LEIRÓS AND R. RAMHORST

Barcelona

Congreso; 34th Annual Meeting of ESHRE; 2018

ESRHE

Study question: Could the decidualization program of endometrial cells induce a sterile inflammatory response through the induction of reticular stress (RS) and unfolded protein response (UPR)? Summary answer: Decidualized cells display a physiological RS and UPR that induce the inflammasome activation generating a sterile inflammatory response with production of IL-1b. What is known already: Embryo implantation in humans requires the generation of a sterile inflammatory response for blastocyst invasion; however, it is still unclear how it could be induced. During decidualization, endometrial stromal cells suffer RS and trigger UPR, allowing them to expand their endoplasmic reticulum and the production of immunomodulators. This physiological RS generates the activation of sensing proteins, which induces kinase/Rnase-TXNIP expression, activating the inflammasome. This multiprotein system allows caspase-1 activation, which catalyzes the cleavage of inactive pro-form IL-1b to secretory mature IL-1b. This highly pro-inflammatory cytokine isassociated with pro-implantatory effects. The sterile inflammatory response should be later controlled in favor of a tolerogenic microenvironment. Study design, size, duration: Human endometrial stromal cell line (HESC) was decidualized or not with medroxiprogesterone+dbcAMP during 8 days, and was used to evaluate RS and UPR. An in vitro implantation model based on co-culture of blastocyst-like spheroids (BLS) from Swan-71 trophoblast cells line over decidualized-HESC cells was used to evaluate decidual functionality. Finally, we evaluated the UPR-inflammatory pathway in endometrial biopsies obtained from fertile women, with recurrent spontaneous abortions (RSA) or with in vitro fertilization failures (RIF).Participants/materials, setting, methods: RS sensors (IRE1, ATF6 and PERK) and UPR markers (sXBP1 and CHOP) were evaluated by RT-qPCR. Inflammasome activation was measured by TXNIP and NLRP3 expression. Caspase-1 activity was evaluated by the fluorescent inhibitor probe FAM-Flica and IL-1b production by FACS analysis, ELISA and Western Blot. Assays were performed also in the absence/presence ofSTF083010 (an Ire1α inhibitor) and Thapsigargin, Tg (RS-inducer). These pahtways were evaluated in endometrial samples from RSA, RIF and fertile women by RT-qPCR.Main results and the role of chance: The decidualized cells (Dec) increased the expression of RS-sensors (ATF6, PERK and IRE1) and UPR markers (sXBP1 and CHOP) in comparison with non-Dec cells. Tg-treated non-Dec cells was used as RS positive control . Then, we evaluated the UPR inflammatory pathway, which activates the inflammasome. Interestingly, while TXNIP expression remains unchanged, we oberved an increased NLRP3 expression in Dec cells compared with non-Dec cells, while STF083010 prevented this increase. Downstream, we detected increased levels of active caspase-1 on Dec cells compared with non-Dec by employing the fluorescent inhibitor probe FAM-Flica Caspase-1, suggesting the NLRP3 inflammasome activation. According to this, decidualized cells significantly increased IL-1b protein expression compared with non dec cells. This multiprotein complex increases IL-1b production in Dec cells. In fact, the inhibition of IRE1α-UPR by STF083010 treatment prevented IL-1b increase in Dec cells and decreased the invasion index evaluated in the in vitro model of implantation, suggesting that the induction of IL-1b is partially dependent of a RS-UPR process. Finally, the present results were validated using endometrial samples. We found an increase expression of IRE1α, sXBP1, TXNIP, NLRP3 and lL-1b on RSA-endometrial biopsies in comparison with fertile women; while biopsies from RIF patients showed a significantly lower expression than fertile women. Large scale data: N/ALimitations, reasons for caution: The present results were obtained using immortalized cell lines and in vitro decidualization and implantation models. Even the results were validated in endometrial samples from fertile women, recurrent spontaneous abortions and in recurrent in vitro fertilization failures, further studies are necessary to elucidate whether these mechanisms operate similarly in vivo.Wider implications of the findings: The present results suggest that human decidualization process is accompained by a physiological RS/UPR, which induces a physiological and sterile inflammatory response associated with an increase of IL-1b production. These processes were differentially affected in RSA and RIF patients in comparison with fertile women, suggesting their relevance on reproductive pathologies.Study funding and competing interest(s):This work was funded by the National Agency of Sciences and Technology ANPCyT(PICT 2013-1632 to RR and 2014-0657 to CPL), and University of Buenos Aires (UBACyT 20020130100040BA to CPL and UBACyT 20020090200034 to RR). The authors have no conflicts of interest to disclose