Immunomodulatory molecules are released from the first trimester and term placenta via exosomes

Abstract

The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular co-localization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 h. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment.

Figure 1. B7-H1 colocalizes with CD63, a marker of late endosomes, in the placenta

First trimester (A–F) and term (G–L) placentas were subjected to immunofluorescence staining followed by confocal microscopy using mouse anti-human B7-H1 (A, D, G, J, M) together with mouse anti-human-CD63 (B, E, H, K, O). Secondary anti-mouse antibodies conjugated with Alexafluor 594 (red) and Alexafluor 488 (green) were used sequentially following each primary antibody. DAPI-counterstained nuclei are shown in blue. Controls included omission of anti-CD63 (M) or anti-B7-H1 (O) primary antibodies together with inclusion of both fluorescently labeled secondary antibodies. Mouse IgG1 was used as a negative control (P). Arrowheads indicate double immunofluorescence (yellow) near the apical membrane of the syncytiotrophoblast; arrows indicate intracellular B7-H1 staining not associated with CD63.

Figure 2. Immunomodulatory proteins are associated with exosomes secreted from first trimester explant cultures

(A) Transmission electron micrograph of apical surface of syncytiotrophoblast of a first trimester placenta. The left side of the image shows microvillus projections of the syncytiotrophoblast; asterisk, multivesicular body; arrows, intralumenal vesicles. (B – D) 24-hour first trimester explant supernatants were ultracentrifuged, and pellets subjected to electron microscopy (B, C) or Western blot analysis (D). Arrows in C demarcate exosomes; arrowheads show contaminating microvesicles. Numbers on left of (D) represent molecular weights (KDa).

Figure 3. Immunomodulators sediment to the density of exosomes

Pellets derived from ultracentrifugation of supernatant from first trimester placental explants were layered over a sucrose gradient, and fractions were subjected to western blot analysis (A) and electron microscopy (B). (B) represents an image of exosomes collected from the 1.152 g/L fraction.

Figure 4. Exosomes lack β2-microglobulin-associated class I HLA

Whole cell lysates of peripheral blood mononuclear cells (PBMC), first trimester explant-derived exosomes, and whole lysate of first trimester villous tissues were subjected to western blot using the conformation-dependent antibody, W6/32, the HLA-G-specific antibody, MEM-G/1, anti-CD63. Blots for W6/32 and CD63 were performed under non-denaturing electrophoresis conditions; blots for HLA-G and LAMP1 were performed under denaturing conditions.

Figure 5. Term placental explants secrete exosomes containing immunomodulators

Term explants were cultured for 24 hours, and exosomes were pelleted and subjected to density gradient centrifugation, followed by analysis by western blot analysis. (A) Western blot analysis of pelleted supernatants; (B) electron microscopic image of pelleted material; (C) Western blot analysis of pelleted material following centrifugation over a sucrose gradient.

Figure 6. Cytotrophoblast cells secrete exosomes in culture

Trophoblast cells were cultured for 3 days, at which time supernatants were subjected to sequential centrifugation. (A) Electron micrograph of pelleted vesicles; (B) Western blot of cell lysate (L1, 5 μg; L2, 10 μg) and pelleted vesicles/exosomes (Ex). Numbers on left of (B) represent molecular weights (KDa).

Figure 7. EGF induces differential protein association with exosomes

(A) Cytotrophoblast cells were cultured for 3 days in exosome-free medium (gray shading) in the absence or presence of EGF, or for 6 days in the presence of EGF. In 6 day cultures, EGF-supplemented medium containing standard FBS was replaced with EGF-suppliemented medium containing exosome-free FBS (gray shading). (B) Pelleted supernatants were subjected to Western blot analysis using antibodies specific for the proteins indicated. Asterisks on right side of figure indicate non-specific bands, most likely due to antibodies binding to residual serum albumin (~68 KDa).