Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states

Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells’ inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi.

The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation

Background Lung inflammation and impaired alveolarization are hallmarks of bronchopulmonary dysplasia (BPD). We hypothesize that human amnion epithelial cells (hAECs) are anti-inflammatory and reduce lung injury in preterm lambs born after antenatal exposure to inflammation. Methods Pregnant ewes received either intra-amniotic lipopolysaccharide (LPS, from E.coli 055:B5; 4mg) or saline (Sal) on day 126 of gestation. Lambs were delivered by cesarean section at 128 d gestation (term ~150 d). Lambs received intravenous hAECs (LPS/hAECs: n = 7; 30x106 cells) or equivalent volumes of saline (LPS/Sal, n = 10; or Sal/Sal, n = 9) immediately after birth. Respiratory support was gradually de-escalated, aimed at early weaning from mechanical ventilation towards unassisted respiration. Lung tissue was collected 1 week after birth. Lung morphology was assessed and mRNA levels for inflammatory mediators were measured. Results Respiratory support required by LPS/hAEC lambs was not different to Sal/Sal or LPS/Sal lambs. Lung tissue:airspace ratio was lower in the LPS/Sal compared to Sal/Sal lambs (P<0.05), but not LPS/hAEC lambs. LPS/hAEC lambs tended to have increased septation in their lungs versus LPS/Sal (P = 0.08). Expression of inflammatory cytokines was highest in LPS/hAECs lambs. Conclusions Postnatal administration of a single dose of hAECs stimulates a pulmonary immune response without changing ventilator requirements in preterm lambs born after intrauterine inflammation.

Amnion epithelial cells are an effective source of factor H and prevent kidney complement deposition in factor H-deficient mice

Complement factor H (FH) is the main plasma regulator of the alternative pathway of complement. Genetic and acquired abnormalities in FH cause uncontrolled complement activation amplifying, with the consequent accumulation of complement components on the renal glomeruli. This leads to conditions such as C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS). There is no effective therapy for these diseases. Half of the patients progress to end-stage renal disease and the condition recurs frequently in transplanted kidneys. Combined liver/kidney transplantation is a valid option for these patients, but the risks of the procedure and donor organ shortages hamper its clinical application. Therefore, there is an urgent need for alternative strategies for providing a normal FH supply. Human amnion epithelial cells (hAEC) have stem cell characteristics, including the capability to differentiate into hepatocyte-like cells in vivo.Here, we administered hAEC into the livers of newborn Cfh−/− mice, which spontaneously developed glomerular complement deposition and renal lesions resembling human C3G. hAEC engrafted at low levels in the livers of Cfh−/− mice and produced sufficient human FH to prevent complement activation and glomerular C3 and C9 deposition. However, long-term engraftment was not achieved, and eventually hAEC elicited a humoral immune response in immunocompetent Cfh−/− mice.hAEC cell therapy could be a valuable therapeutic option for patients undergoing kidney transplantation in whom post-transplant immunosuppression may protect allogeneic hAEC from rejection, while allogeneic cells provide normal FH to prevent disease recurrence.

Regulating cell fate of human amnion epithelial cells using natural compounds: an example of enhanced neural and pigment differentiation by 3,4,5-tri-O-caffeoylquinic acid

Over the past years, Human Amnion Epithelial Cells (hAECs), a placental stem cell, are gaining higher attention from the scientific community as they showed several advantages over other types of stem cells, including availability, easy accessibility, reduced rejection rate, non-tumorigenicity, and minimal legal constraint. Recently, natural compounds are used to stimulate stem cell differentiation and proliferation and to enhance their disease-treating potential. A polyphenolic compound 3,4,5-Tri-O-Caffeoylquinic Acid (TCQA) has been previously reported to induce human neural stem cell differentiation and may affect melanocyte stem cell differentiation as well. In this study, TCQA was tested on 3D cultured hAECs after seven days of treatment, and then, microarray gene expression profiling was conducted of TCQA-treated and untreated control cells on day 0 and day 7. Analyses revealed that TCQA treatment significantly enriched pigment and neural cells sets; besides, genes linked with neurogenesis, oxidation–reduction process, epidermal development, and metabolism were positively regulated. Interestingly, TCQA stimulated cell cycle arrest-related pathways and differentiation signaling. On the other hand, TCQA decreased interleukins and cytokines expression and this due to its anti-inflammatory properties as a polyphenolic compound. Results were validated to highlight the main activities of TCQA on hAECs, including differentiation, cell cycle arrest, and anti-inflammatory. This study highlights the important role of hAECs in regenerative medicine and the use of natural compounds to regulate their fate.

Inflammation, but not infection, induces EMT in human amnion epithelial cells

A non-reversible state of epithelial to mesenchymal transition (EMT) at term accumulates proinflammatory mesenchymal cells and predisposes fetal membrane to weakening prior to delivery at term. We investigated the induction of EMT in amnion epithelial cells (AEC) in response to inflammation and infection associated with spontaneous preterm birth (SPTB). For this, membranes from SPTB were screened for EMT markers. Primary AEC in culture were treated with TNF-α (10 and 50 ng/mL) and LPS (50 and 100 ng/mL) for 72 h. Cell shape index (SI) was determined based on morphological shift (microscopy followed by ImageJ software analysis). Immunocytochemistry and Western blot assessed changes in epithelial markers (cytokeratin-18 and E-cadherin) and mesenchymal markers (vimentin and N-cadherin). Involvement of transforming growth factor beta (TGF-β) in EMT induction and EMT associated inflammation was tested using specific markers (Western blot) and by measuring MMP9 (ELISA), respectively. We report that PTB is associated with fetal membrane EMT. TNF-α produced dose- and time-dependent induction of EMT; within 24 h by 50 ng/mL and after 72 h by 10 ng/mL. AEC showed mesenchymal morphology, lower E-cadherin, higher vimentin and N-cadherin and higher MMP9 compared to control. TNF-α-induced EMT was not associated with canonical TGF-β pathway. LPS, regardless of dose or time, did not induce EMT in AEC. We conclude that PTB with intact membranes is associated with EMT. Our data suggest that inflammation, but not infection, is associated with non-canonical activation of EMT and inflammation that can predispose membrane to undergo weakening.

Human Amnion Epithelial Cells Impair T Cell Proliferation: The Role of HLA-G and HLA-E Molecules

The immunoprivilege status characteristic of human amnion epithelial cells (hAECs) has been recently highlighted in the context of xenogenic transplantation. However, the mechanism(s) involved in such regulatory functions have been so far only partially been clarified. Here, we have analyzed the expression of HLA-Ib molecules in isolated hAEC obtained from full term placentae. Moreover, we asked whether these molecules are involved in the immunoregulatory functions of hAEC. Human amnion-derived cells expressed surface HLA-G and HLA-F at high levels, whereas the commonly expressed HLA-E molecule has been measured at a very low level or null on freshly isolated cells. HLA-Ib molecules can be expressed as membrane-bound and soluble forms, and in all hAEC batches analyzed we measured high levels of sHLA-G and sHLA-E when hAEC were maintained in culture, and such a release was time-dependent. Moreover, HLA-G was present in extracellular vesicles (EVs) released by hAEC. hAEC suppressed T cell proliferation in vitro at different hAEC:T cell ratios, as previously reported. Moreover, inhibition of T cell proliferation was partially reverted by pretreating hAEC with anti-HLA-G, anti-HLA-E and anti-β2 microglobulin, thus suggesting that HLA-G and -E molecules are involved in hAEC-mediated suppression of T cell proliferation. Finally, either large-size EV (lsEV) or small-size EV (ssEV) derived from hAEC significantly modulated T-cell proliferation. In conclusion, we have here characterized one of the mechanism(s) underlying immunomodulatory functions of hAEC, related to the expression and release of HLA-Ib molecules.