Ectodysplasin-A mRNA in exosomes released from activated hepatic stellate cells stimulates macrophage response.

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.