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Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1β and IL-17 upregulation in alcoholic hepatitis mice.

ABSTRACT: Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The numbers of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared with control mice. The miRNA (miR)-seq analysis detected 20 upregulated and 4 downregulated miRNAs (P < 0.001-0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced α-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7 and Nr1d2 genes, which were downregulated in HSCs from the AH mice, were predicted targets of 20 miRs upregulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, indeed repressed Nr1d2, the quiescent HSC marker. AH-HC-EVs were also enriched with organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1β and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting the quiescent HSC transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1β and IL-17 by HMs. KEY MESSAGES: • Circulating EVs and HC-EVs were increased in AH mice compared with control mice • AH-HC-EVs were enriched in miRNAs, organelle proteins, and mitochondrial DNA • AH-HC-EVs increased cytokine production by AH-HMs in a TLR9-dependent manner.

SUBMITTER: Eguchi A

PROVIDER: S-EPMC7810220 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Neuro-immune interaction during development is strongly implicated in the pathogenesis of neurodevelopmental disorders, but the mechanisms that cause neuronal circuit dysregulation are not well understood. We performed in vivo imaging of the developing retinotectal system in the larval zebrafish to characterize the effects of immune system activation on refinement of an archetypal sensory processing circuit. Acute inflammatory insult induced hyper-dynamic remodeling of developing retinal axons in larval fish and increased axon arbor elaboration over days. Using calcium imaging in GCaMP6s transgenic fish we showed that these morphological changes were accompanied by a shift toward decreased visual acuity in tectal cells. This finding was supported by poorer performance in a visually guided behavioral task. We further found that the pro-inflammatory cytokine, interleukin-1β (IL-1β) is upregulated by the inflammatory insult, and that down-regulation of IL-1β abrogated the effects of inflammation on axonal dynamics and growth. Moreover, baseline branching of the RGC arbors in IL-1β morphant animals was significantly different from that in control larvae, and their performance in a predation assay was impaired, indicating a role for this cytokine in normal neuronal development. This work establishes a simple and powerful non-mammalian model of developmental immune activation and demonstrates a role for IL-1β in mediating the pathological effects of inflammation on neuronal circuit development.SIGNIFICANCE STATEMENTMaternal immune activation (MIA) can increase the risk of neurodevelopmental disorders in offspring, however the mechanisms involved are not fully understood. Using a non-mammalian vertebrate model of developmental immune activation, we show that even brief activation of inflammatory pathways has immediate and long-term effects on the arborization of axons, and that these morphological changes have functional and behavioral consequences. Finally, we show that the pro-inflammatory cytokine IL-1β plays an essential role in both the effects of inflammation on circuit formation and normal axonal development. Our data add to a growing body of evidence supporting epidemiological studies linking immune activation to neurodevelopmental disorders, and help shed light on the molecular and cellular processes that contribute to the etiology of these disorders.

Project description:AimTo investigate over-expression of Osteopontin (OPN) pathway expression and mechanisms of action in human alcoholic liver disease (ALD), in vivo and in vitro acute alcohol models.MethodsOPN pathway was evaluated in livers from patients with progressive stages of human ALD and serum from drinkers with and without liver cirrhosis. In vitro stellate LX2 cells exposed to acute alcohol and in vivo in acute alcoholic steatosis mouse models were also investigated for OPN pathway expression and function. WT and OPN(-/-) mice were administered an acute dose of alcohol and extent of liver injury was examined by histopathology and liver biochemistry after 16-24 h. The causative role of OPN was studied in OPN knockout animals and in vitro in stellate LX2 cells, utilizing siRNA, aptamer and neutralizing antibodies to block OPN and OPN pathway. OPN pathway expression and downstream functional consequences were measured for signaling by Western blotting, plasmin activation by spectrophotometric assays and cell migration by confocal imaging and quantitation.ResultsOPN expression positively correlated with disease severity in patients with progressive stages of ALD. In vivo, associated with alcoholic steatosis, a single dose of acute alcohol significantly increased hepatic OPN mRNA and protein, and a cleaved OPN form in a dose dependent manner. OPN mRNA and secreted OPN also increased in parallel with activation of LX2 stellate cells within 4 h of a single dose of alcohol. Expression of OPN receptors, αvβ3-integrin and CD44, increased in human ALD, and in vivo and in vitro with alcohol administration. This was accompanied by downstream phosphorylation of Akt and Erk, increased mRNA expression of several fibrogenesis, fibrinolysis and extracellular matrix pathway genes, plasmin activation and hepatic stellate cell (HSC) migration. Inhibition of OPN and OPN-receptor mediated signaling partially inhibited alcohol-induced HSC activation, plasmin activity and cell migration.ConclusionOPN is a key mediator of the alcohol-induced effects on hepatic stellate cell functions and liver fibrogenesis.

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Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1β and IL-17 upregulation in alcoholic hepatitis mice.

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