Forskningsrapport från Ungern

Blogger har fått mejl från 2 olika läsare som tipsar om en alldeles ny forskningsartikel.
7 ungerska forskare har alltså fått sina studier publicerade i International Journal of Molecular Sciences. 
Det ser ut som studien fick offentlighet först igår söndag.

Studien handlar om hur kvinnans immunsystem agerar/fungerar vid en graviditet.Det då ett foster har risk för att uppfattas som ett hot mot bärarens immunsystem.Jag förmodar att det har att göra med risken för avstötning och missfall.
Unravelling the Role of Trophoblastic-Derived Extracellular Vesicles in Regulatory T Cell Differentiation

Received: 21 June 2019 / Accepted: 11 July 2019 / Published: 14 July 2019

Abstract

Regulatory T cells (T_reg) are mandatory elements in the maintenance of human pregnancy, but their de novo differentiation has not been completely exposed. HSPE1 chaperone expressing trophoblast cells may have a role in it. Trophoblast-derived extracellular vesicles (EVs), either at the feto–maternal interface or in circulation, target CD4⁺ T cells. We hypothesized that HSPE1-associated trophoblastic cell line (BeWo)-derived EVs are active mediators of T_reg cell differentiation. We proved at first that recombinant HSPE1 promote human T_reg cell differentiation in vitro. Developing a CRISPR-Cas9 based HSPE1 knockout BeWo cell line we could also demonstrate, that EV-associated HSPE1 induces T_reg development. Next-generation sequencing of miRNA cargo of BeWo-EVs characterized the regulatory processes of T_reg polarization. By the use of single-cell transcriptomics analysis, seven T_reg cell subtypes were distinguished and we demonstrated for the first time that the expression level of HSPE1 was T_reg subtype dependent, and CAPG expression is characteristic to memory phenotype of T cells. Our data indicate that HSPE1 and CAPG may be used as markers for identification of T_reg subtypes. Our results suggest, that trophoblastic-derived iEVs-associated HSPE1 and miRNA cargo have an important role in T_reg cell expansion in vitro and HSPE1 is a useful marker of T_reg subtype characterization.

Introduction

During human pregnancy the maternal immune system has to fine balance itself, in order to develop and maintain immune tolerance toward the semi-allograft fetus. An ongoing equilibrium between proinflammatory and anti-inflammatory stimuli characterize the immune system, and a tolerogenic environment is essential for the maintenance of pregnancy . Immune suppressive T_reg cells are crucial players of peripheral tolerance. T_reg cells are found at high levels in decidual tissues, and recently it has been described that in vitro co-culture of extravillous trophoblast cells with blood derived CD4⁺ T cells induces T_reg cell differentiation. Nevertheless, it has not been clearly demonstrated what mechanisms are involved in the de novo differentiation of T_reg cells at the feto–maternal interface. Although the generation of peripheral T_reg cells is less clearly understood , intercellular communication may have an important role in it. One of the most dynamic forms of intercellular communication is mediated by extracellular vesicles (EVs). EVs released by cells are heterogeneous in size, the two most well-studied subtypes are the intermediate-sized EVs (size range 150–800 nm, iEVs) and small EVs (size range 35–150 nm, sEVs). The placenta is an abundant and transient source of EVs. On the other hand, the key feature of the protective environment is particularly realized at the placental level via the establishment and maintenance of tolerance.

Trophoblast cells highly express the HSPE1 protein. The main annotated role of HSPE1 is the chaperonin mediated protein folding together with Hsp60 . It has also been suggested that HSPE1 may be critical in the suppression of T cell activation. In myocytes, it has been also described that HSPE1 inhibits the proapoptotic activity of cells and shifts the cell fate balance toward survival. 

It has also been proposed that HSPE1 is selectively released from proliferating cells and is an active player of cell signalling network. Recombinant HSPE1 selectively binds to human CD4⁺ T cells in vitro and can induce T_reg differentiation in mouse animal model. Besides proteins, miRNAs are the most important molecular cargo of EVs. miRNAs dynamically regulate the expression of gene networks (1) and contribute to the function of cell differentiation at the posttranscriptional level . 

 EV-associated miRNome is regarded as one of the most promising clinical biomarker for diagnosis, prognosis, and therapeutic options . On the basis of these data, we hypothesized that trophoblast-derived EV-associated HSPE1 and miRNA cargo specify the generation and heterogeneity of T_reg cells at the feto–maternal interface.

Bland material och metodik klipper jag som vanligt in enbart PHI-relaterat.

 

Materials and Methods

Holomicroscopy of T_reg Cells

CD25^hi+ CD127^lo+ sorted T_reg cells were characterized by holomicroscopy (HoloMonitor M4 Phase Holographic Imaging, Lund, Sweden). Five thousand T_reg cells were resuspended in 500 µL of cell culture media and seeded on µ-Slide 8-Well with ibiTreat (Ibidi, Martinsried, Germany) and incubated for 30 min to allow cell adhesion. 

Cell migration (the shortest direct distance from the starting point to the end point (μm)) and motility (the actual way traveled from the starting point to the end point (μm)) were monitored for 3 h with 60 sec time-lapse intervals. 

Time-lapse automatic background thresholding method was used with the minimum error sets algorithm (adjustment = 128) for evaluation of images. 

The total number of evaluated images were 180 and the number of evaluated cells per image was 10.
For manual cell identification, objects of the marginal zone were eliminated. 

HoloStudio M4 2.5 program was used to analyze data.

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