Human amnion mesenchymal cells are pro-inflammatory when activated by the Toll-like receptor 2/6 ligand, macrophage-activating lipoprotein-2

Abstract

Introduction Infection accounts for over 40% of preterm premature rupture of the fetal membranes (PPROM), a major cause of preterm birth. Toll-like receptors (TLR) play key roles in pathogen surveillance but their expression and function in amnion mesenchymal cells (AMC) is unclear. The aims of this study were to determine the expression of all TLR isoforms and the effect of macrophage-activating lipoprotein-2 (MALP-2), derived from a common pathogen involved in PPROM, on human AMC. Methods AMC were isolated from normal, term, amnion from repeat caesarean section. Semi-quantitative RT-PCR, immunocytochemistry, immunohistochemistry and western blotting were used to detect TLR isoform expression. Immunocytochemistry of NF-κB p65, pro-inflammatory cytokine secretion (ELISA), MTT assay, LDH assay, immunoblotting of cytosolic cytochrome c and cleaved caspase-3, and expression of 84 microRNAs by Qiagen miRNA PCR array were used to determine the functional effect of MALP-2 on AMC. Results TLR1-10 was detected in AMC, and protein expression of TLR2, 4, and 6 were confirmed. MALP-2 induced nuclear translocation of p65, reaching significance after 45 minutes (ANOVA, P < 0.05). MALP-2 did not cause apoptosis but did lead to significant secretion of IL-4, IL-6, and IL-8 (P < 0.05, 0.01, 0.001, respectively) and significant changes in miRNA-320a and miRNA-18a (P < 0.05). Discussion These results suggest that AMC elicit a pro-inflammatory response following stimulation with the known TLR2/6 ligand MALP-2. This data supports the idea that AMC express the innate immune system receptors that could help with immune surveillance during infection and contribute to inflammatory responses that lead to PPROM.

Organ-On-Chip Technology: The Future of Feto-Maternal Interface Research?

“…Though this brings in patient-to-patient variability ( Table 1 ), these approaches maintain some of the in vivo characteristics such as cytoskeletal organization ( Menon et al, 2018 ; Richardson et al, 2018b , 2020b ), endocrine and paracrine signaling ( Myatt and Sun, 2010 ; Behnia et al, 2015 ), inflammatory responses ( Menon et al, 2009 ; Noda-Nicolau et al, 2016 ), as well as immune regulatory factors ( Fortunato et al, 1998 , 2001 ). Protocols documenting amnion (i.e., AEC and AMC) cell isolation techniques are well established ( Kendal-Wright, 2007 ; Menon et al, 2013 ; Sato et al, 2016 ; Jin et al, 2018 ). However, although it is not impossible to isolate and culture primary chorion mesenchymal and trophoblast cells (CMC and CT) [99, 108], due to many in vitro challenges (isolation, culture conditions, passage-related issues, and transition properties), researchers have turned to use immortalized placenta-based trophoblast cells (i.e., BEWO and JEG-3) derived from carcinomas to replicate this layer [109].…”

“…Two-dimensional (2D) single cell type culture experiments are most easy to run and low cost, and thus broadly utilized (Kendal-Wright, 2007;Menon et al, 2013;Meng et al, 2016;Sato et al, 2016;Feng et al, 2018b;Hadley et al, 2018;Jin et al, 2018). However, regardless of the cell origin (i.e., primary or immortalized) or cell layer (i.e., amnion or chorion), the major drawback is the limitations stemming from studying only a small part of the whole organ.…”

“…The placenta plays a critical role in maintaining pregnancy by regulating maternal metabolism, endocrine and immune functions in addition to providing blood flow [arteries (blue) and veins (red)], nutrients, and oxygen to the fetus, while removing waste products such as carbon dioxide ( Jabareen et al, 2009 ; Mauri et al, 2013 ; Edey et al, 2018 ) ( Figures 2A,B ). The fetal membrane, which surrounds the baby throughout gestation, provides essential immune, endocrine, and mechanical functions ( Figures 2B,C ) that maintain pregnancy ( Jabareen et al, 2009 ; Boldenow et al, 2013 ; Mauri et al, 2013 , 2015 ; Perrini et al, 2015 ; Menon, 2016 ; Menon et al, 2016 , 2017 ; Sato et al, 2016 ; Edey et al, 2018 ; Shah et al, 2019 ). They are comprised of two epithelial layers, the amnion (blue) and chorion (yellow), separated by an ECM containing mesenchymal cells (purple).…”

Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

“…Human AMCs were isolated from the fetal membranes as per the protocol described previously by Sato et al [ 77 ] with slight modifications. Briefly, AECs were removed from the collected fetal membranes by incubation with 0.05% trypsin/EDTA (Corning, Corning, NY, USA) for 1 h in a 37 °C water bath.…”

Toll-Like Receptors: The Key of Immunotherapy in MSCs

“…Nevertheless, hTM-SCs were only responsive to TLR4 as displayed by the significant changes in their cytokine profiles [133]. Macrophage-activating ligand-2 (MALP-2), an agonist of TLR6, as well as its heterodimer partner TLR2, initiated the activation of NF-κβ pathway leading AMC to obtain a pro-inflammatory profile by highly secreting cytokines as IL-4, IL-8 and IL-6 [134].…”