Human Amnion Epithelial Cells (AECs) Respond to the FSL-1 Lipopeptide by Engaging the NLRP7 Inflammasome

Abstract

Context and Objectives: Inflammation is the leading mechanism involved in both physiological and pathological rupture of fetal membranes. Our aim was to obtain a better characterization of the inflammasome-dependent inflammation processes in these tissues, with a particular focus on the nucleotide-binding oligomerization domain (NOD)-like receptor, pyrin domain containing protein 7 (NLRP7) inflammasome. Methods: The presence of NLRP7 inflammasome actors [NLRP7, apoptosis-associated speck-like protein containing a CARD domain (ASC), and caspase-1] was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) in human amnion and choriodecidua at the three trimesters and at term. The protein concentrations were then determined by enzyme-linked immunosorbent assay in term tissues, with or without labor. The presence of Mycoplasma salivarium and Mycoplasma fermentans in human fetal membranes was investigated using a PCR approach. Human amnion epithelial cells (AECs) were treated for 4 or 20 h with fibroblast-stimulating lipopeptide-1 (FSL-1), a M. salivarium-derived ligand. Transcripts and proteins quantity was then measured by RT-quantitative PCR and Western blotting, respectively. NLRP7 and ASC colocalization was confirmed by immunofluorescence. Western blots allowed analysis of pro-caspase-1 and gasdermin D cleavage. Results: NLRP7, ASC, and caspase-1 transcripts were expressed in both sheets of human fetal membranes during all pregnancy stages, but only ASC protein expression was increased with labor. In addition, M. salivarium and M. fermentans were detected for the first time in human fetal membranes. NLRP7 and caspase-1 transcripts, as well as NLRP7, ASC, and pro-caspase-1 protein levels, were increased in FSL-1-treated AECs. The NLRP7 inflammasome assembled around the nucleus, and pro-caspase-1 and gasdermin D were cleaved into their mature forms after FSL-1 stimulation. Conclusion: Two new mycoplasmas, M. salivarium and M. fermentans, were identified in human fetal membranes, and a lipopeptide derived from M. salivarium was found to induce NLRP7 inflammasome formation in AECs.

Characterization of RAGE and CK2 Expressions in Human Fetal Membranes

“…Primary amniocyte cells were collected from the living tissue of the amnion after trypsination and were cultured in vitro on six-well plates coated with bovine collagen type I/III (StemCell Technologies, Grenoble, France), as described previously [ 46 , 47 ]. The primary amniocytes in the TIL and TNL groups were respectively collected from fresh fetal membranes: (38–41 WG) and (39–40 WG).…”

Pathophysiological Implication of Pattern Recognition Receptors in Fetal Membranes Rupture: RAGE and NLRP Inflammasome

“…The presence of the three NLRP7 inflammasome actors (ASC, caspase 1 and NLRP7) has been characterized in the amnion and chorion but also in primary amniotic epithelial cells (AECs) that were collected from the FM of women with term deliveries. It suggests that this NLRP7 inflammasome can assemble in the FM at all stages of pregnancy [ 157 ]. According to the literature, only one ligand is known and has been used to specifically activate the NLRP7 inflammasome: fibroblast stimulating lipopeptide (FSL-1) [ 158 ].…”

“…Numerous studies have used FSL-1 on FM, amnion cells or myometrial cells [ 22 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 ]; they allowed for the characterization of the signaling pathway induced by FSL-1, and they demonstrated an induction of the synthesis of pro-inflammatory mediators. The NLRP7 inflammasome, though specifically activated by this ligand, has been studied in the FM context in only one study [ 157 ].…”

“…Thus, Lavergne et al demonstrated that AECs can form a functional NLRP7 inflammasome. More interestingly, the quantification of the expression of these three actors in the FM revealed an overexpression of ASC (in both layers of FM) at term with labor compared with term FM without labor [ 157 ]. This observation confirms the study of Gomez-Lopez et al , who described an increase of ASC/Caspase-1 complexes in the FM with labor [ 138 ].…”