Instituto de Genética Humana, Facultad de Medicina, Pontificia Universidad Javeriana, Bogotá, Colombia.
Viral Immunol. 2010 Dec;23(6):595-608. doi: 10.1089/vim.2009.0113.
Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and "danger signals" released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-β1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-β, because they were reversed by treatment of the T cells with the TGF-β-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity.
轮状病毒（RV）主要在肠道上皮细胞（IEC）中复制，这些细胞释放的“危险信号”可能调节病毒免疫。我们最近表明，感染恒河猴 RV 的人肠道上皮细胞模型（Caco-2 细胞）释放一组非炎症性免疫调节剂，包括热休克蛋白（HSPs）和 TGF-β1。在这里，我们表明这两种蛋白部分与从超滤浓缩的 Caco-2 上清液过滤得到的膜囊泡（MV）相关联而被释放。这些 MV 表达外体（CD63 等）的标志物，但不表达内质网（ER）或核的标志物。与未感染细胞相比，感染 RV 的细胞释放与 MV 相关的蛋白数量更多。VP6 与存在于这些 MV 中的 CD63 共同免疫沉淀，并且 VP6 在感染 RV 的细胞中与 CD63 共定位，表明该病毒蛋白与 MV 相关联，并且这种关联发生在细胞内。分析了来自因或不因 RV 引起肠胃炎的 36 名儿童粪便样本中 MV 制备物中的 CD63。在 8 份来自 RV 感染儿童的粪便样本中，VP6 与 CD63 共同免疫沉淀，这表明这些 MV 是由 RV 感染细胞在体内释放的。此外，与未感染细胞相比，来自 RV 感染细胞的包含 MV 的分数更能诱导 CD4+T 细胞死亡并抑制其增殖。这些作用部分归因于 TGF-β，因为用 TGF-β 受体抑制剂 ALK5i 处理 T 细胞可逆转这些作用。来自 RV 感染和未感染细胞的 MV 是异质的，具有描述外体的形态和典型的浮密度（在 1.10 到 1.18g/mL 之间），以及更致密的囊泡（>1.24g/mL）。与未感染细胞相比，来自 RV 感染细胞的这两种类型的 MV 更有效地抑制 T 细胞功能。我们提出，IEC 中 RV 的感染释放了调节病毒免疫的 MV。
