Microbiota-Associated HAF-EVs Regulate Monocytes by Triggering or Inhibiting Inflammasome Activation.

In pregnancy, human amniotic fluid extracellular vesicles (HAF-EVs) exert anti-inflammatory effects on T cells and on monocytes, supporting their immunoregulatory roles. The specific mechanisms are still not completely defined. The aim of this study was to investigate the ability of HAF-EVs, isolated from pregnant women who underwent amniocentesis and purified by gradient ultracentrifugation, to affect inflammasome activation in the human monocytes. Proteomic studies revealed that HAF-EV samples expressed several immunoregulatory molecules as well as small amounts of endotoxin. Surprisingly, metagenomic analysis shows the presence of specific bacterial strain variants associated with HAF-EVs as potential sources of the endotoxin. Remarkably, we showed that a single treatment of THP-1 cells with HAF-EVs triggered inflammasome activation, whereas the same treatment followed by LPS and ATP sensitization prevented inflammasome activation, a pathway resembling monocyte refractories. A bioinformatics analysis of microbiota-HAF-EVs functional pathways confirmed the presence of enzymes for endotoxin biosynthesis as well as others associated with immunoregulatory functions. Overall, these data suggest that HAF-EVs could serve as a source of the isolation of a specific microbiota during early pregnancy. Moreover, HAF-EVs could act as a novel system to balance immune training and tolerance by modulating the inflammasome in monocytes or other cells.

Amniotic fluid stem cell-derived extracellular vesicles are independent metabolic units capable of modulating inflammasome activation in THP-1 cells.

An immunoregulatory role of stem cells, often mediated by their secretome, has been claimed by several studies. Stem cell-derived extracellular vesicles (EVs) are crucial components of the secretome. EVs, a heterogeneous group of membranous vesicles released by many cell types into the extracellular space, are now considered as an additional mechanism for intercellular communication. In this study, we aimed at investigating whether human amniotic stem cell-derived extracellular vesicles (HASC-EVs) were able to interfere with inflammasome activation in the THP-1 cell line. Two subsets of HASC-EVs were collected by sequential centrifugation, namely HASC-P10 and HASC-P100. We demonstrated that HASC-EVs were neither internalized into nor undertake a direct interaction with THP-1 cells. We showed that HASC-P10 and P100 were able to intrinsically produce ATP, which was further converted to adenosine by 5'-nucleotidase (CD73) and ectonucleoside triphosphate diphosphohydrolase-1 (CD39). We found that THP-1 cells conditioned with both types of HASC-EVs failed to activate the NLRP3/caspase-1/inflammasome platform in response to LPS and ATP treatment by a mechanism involving A2a adenosine receptor activation. These results support a role for HASC-EVs as independent metabolic units capable of modifying the cellular functions, leading to anti-inflammatory effects in monocytic cells.

Tumor protein D52 (TPD52): a novel B-cell/plasma-cell molecule with unique expression pattern and Ca(2+)-dependent association with annexin VI.

We generated a murine monoclonal antibody (B28p) detecting an antigenic determinant shared by the immunoglobulin superfamily receptor translocation-associated 1 (IRTA1) receptor (the immunogen used to raise B28p) and an unrelated 28-kDa protein that was subsequently subjected to extensive characterization. The expression of the 28-kDa protein in normal lymphohematopoietic tissues was restricted to B cells and plasma cells and clearly differed from that expected for IRTA1 (selectively expressed by mucosa-associated lymphoid tissue [MALT] marginal zone B cells). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)/mass-spectrometry analysis identified the 28-kDa protein as human tumor protein D52 (TPD52), whose expression had been previously described only in normal and neoplastic epithelia. Specific B28p reactivity with TPD52 was confirmed by immunostaining/immunoblotting of TPD52-transfected cells. TPD52 expression pattern in normal and neoplastic B cells was unique. In fact, unlike other B-cell molecules (paired box 5 [PAX5], CD19, CD79a, CD20, CD22), which are down-regulated during differentiation from B cells to plasma cells, TPD52 expression reached its maximum levels at the plasma cell stage. In the Thiel myeloma cell line, TPD52 bound to annexin VI in a Ca(2+)-dependent manner, suggesting that these molecules may act in concert to regulate secretory processes in plasma cells, similarly to what was observed in pancreatic acinar cells. Finally, the anti-TPD52 monoclonal antibody served as a valuable tool for the diagnosis of B-cell malignancies.