Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia

TLDR

Exosomes are small extracellular vesicles that accumulate in intrahepatic bile ducts and are thought to interact with cholangiocyte cilia, but direct evidence and physiological relevance remain unclear. This study tested whether biliary exosomes communicate with cholangiocyte cilia to trigger intracellular signaling and alter cell proliferation. Exosomes were isolated from rat bile by differential ultracentrifugation, characterized by electron microscopy, and their effects on ERK1/2 signaling, miR‑15A expression, and proliferation were examined in ciliated versus deciliated cultured cholangiocytes. Bile exosomes (CD63⁺/Tsg101⁺, saucer‑shaped) attached to cilia, reduced pERK/ERK, increased miR‑15A, and suppressed proliferation, effects lost when cilia were removed, indicating exosomes act as signaling nanovesicles via primary cilia.

Abstract

Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic “saucer-shaped” morphology, and specific markers, CD63 and Tsg101. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia.