ABSTRACT
INTRODUCTION: The interaction between activated hepatic stellate cells (aHSCs) and macrophages is central to liver fibrosis development. The cargo contained within aHSC exosomes (aHSC-EXOs) and how aHSC-EXOs affect macrophage function is poorly understood. METHODS: RNA from aHSC-EXOs was separated into small (<200-basepairs) and large (≥200-basepairs) RNA species, transfected into macrophages, and macrophage IL-6 and TNFα mRNA expression and protein secretion measured. Next generation sequencing was performed on EXOs from rat quiescent and aHSCs and human aHSCs. aHSCs were transfected with siRNA against ectodysplasin-A (EDA), EXOs collected, and their effect on macrophage function analyzed. Human cirrhotic liver was analyzed for EDA mRNA expression and compared to non-tumor liver (NTL). RESULTS: Transfection with large RNA from aHSC-EXOs stimulated macrophage IL-6 and TNFα mRNA expression and protein secretion. EDA mRNA was highly expressed in aHSCs and transfection of aHSCs with EDA-siRNA decreased aHSC-EXO EDA mRNA and blunted the effect of aHSC-EXOs on macrophage function (IL-6/TNFα expression and macrophage migration). Human cirrhotic liver exhibited high EDA mRNA compared to NTL. CONCLUSIONS: HSC activation leads to altered EXO mRNA/miRNA profiles with aHSC-EXOs mRNAs exerting a dominant role in altering macrophage function. Ectodysplasin-A mRNA is an important component in aHSC-EXOs in regulating macrophage function.
Subject(s)
Exosomes , Liver Neoplasms , Animals , Ectodysplasins/metabolism , Ectodysplasins/pharmacology , Edar Receptor , Exosomes/metabolism , Hepatic Stellate Cells/metabolism , Humans , Interleukin-6/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/pathology , Macrophages/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rats , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: Hepatic stellate cell (HSC) differentiation/activation is central to liver fibrosis and is innately linked to the immune response to liver injury. Exosomes (EXOs) are important means of communication between cell populations. This study sought to characterize EXO release from HSCs and the effect of HSC-EXOs on macrophage cytokine release/function. METHODS: Liver from a rat fibrosis model was analyzed for EXO expression and localization. Quiescent and culture-activated rat and mouse HSCs and activated human HSCs were analyzed for microRNA expression. Mouse, rat, and human HSCs were culture-activated and EXOs purified from culture medium prior to addition to macrophages, and interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) mRNA and protein measured. The effect of activated HSC-EXOs on macrophage migration was assayed. RESULTS: Activation of rat HSCs led to increased EXO production in vivo, an effect mirrored by in vitro rat HSC culture-activation. Culture activation of mouse and rat HSCs led to altered EXO microRNA profiles, with a similar microRNA profile detected in activated human HSCs. Addition of activated HSC-EXOs to macrophages stimulated IL-6 and TNFα mRNA expression and protein secretion in mouse and human macrophages, but not for rat HSC-EXO-macrophages. Addition of human EXOs to macrophages stimulated migration, effects mirrored by the direct addition of rhIL-6 and rhTNFα. CONCLUSIONS: HSC-EXOs associate with macrophages and stimulate cytokine synthesis-release and macrophage migration. Constructing a comprehensive understanding of EXO interactions between liver cell populations in the setting of inflammation/fibrosis increases the potential for developing new diagnostic/therapeutic approaches.
