Hepatic stellate cell-derived exosomes modulate macrophage inflammatory response.

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.

Adeno-Associated Virus Serotype 2 Vector-Mediated Reintroduction of microRNA-19b Attenuates Hepatic Fibrosis.

Fibrotic liver injury is a significant healthcare burden in the United States. It represents a major cause of morbidity and mortality for which there are no effective Food and Drug Administration-approved treatment strategies. Fibrosis is considered a disruption of the normal wound healing responses mediated by fibroblastic cells, which are triggered and sustained by pro-fibrotic cytokines such as transforming growth factor beta 1 (TGF-ß1). TGF-ß1-mediated trans-differentiation of hepatic stellate cells (HSCs) from quiescent to activated myofibroblasts is a pivotal event in the development of fibrosis. Activation is accompanied by global changes in microRNA (miR) expression. It has been previously reported that miR19b is decreased in activated HSCs and contributes to increased expression of TGF-ß receptor II and connective tissue growth factor, both confirmed targets of miR19b. An adeno-associated virus serotype 2 vector (AAV2) with a miR19b transgene downstream of enhanced green fluorescent protein under the murine collage alpha 1(I) promoter was developed specifically to target HSCs. Male Sprague Dawley rats (250 g) underwent sham or bile-duct ligation (BDL) surgery. Directly after BDL, rats received AAV2-miR19b, AAV2-control, or vehicle normal saline (NS) by portal-vein injection. After 2 weeks, the animals were euthanized, and blood was collected for alanine and aspartate aminotransferase, total and direct bilirubin, and alkaline phosphatase. Tissue was collected for RNA and protein extraction and histology. Fibrosis and measures of hepatic injury were significantly reduced in AAV2-miR19b-treated rats in combination with significant improvements in total and direct bilirubin. Histological analysis of collagen by PicroSirius Red staining revealed a ∼50% reduction compared to AAV2-control or NS-injected animals. Pro-fibrotic markers, smooth-muscle alpha-actin, TGF-ß receptor II, and collagen alpha 2(I) mRNA and protein were significantly decreased compared to AAV2-control and NS groups. AAV2-mediated reintroduction of miR-19b, specifically expressed in HSCs, improved liver function, inhibited fibrosis, and improved measures of hepatic injury in a BDL model.

Processing of miR17-92 Cluster in Hepatic Stellate Cells Promotes Hepatic Fibrogenesis During Alcohol-Induced Injury.

BACKGROUND: Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through hepatic stellate cell (HSC) activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media (TCM) and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood. METHODS: Expression of primary (pri)-miR17-92. and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b, and 92, were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from TCM and plasma of rodent models and patients with alcoholic liver disease (ALD). miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. RESULTS: Profibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased pri-miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD was similar to that seen in a rodent model of alcoholic injury and in vitro. CONCLUSIONS: Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights into the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.