Stimulation of Liver Fibrosis by N2 Neutrophils in Wilson's Disease.

BACKGROUND & AIMS: Wilson's disease is an inherited hepatoneurologic disorder caused by mutations in the copper transporter ATP7B. Liver disease from Wilson's disease is one leading cause of cirrhosis in adolescents. Current copper chelators and zinc salt treatments improve hepatic presentations but frequently worsen neurologic symptoms. In this study, we showed the function and machinery of neutrophil heterogeneity using a zebrafish/murine/cellular model of Wilson's disease. METHODS: We investigated the neutrophil response in atp7b-/- zebrafish by live imaging, movement tracking, and transcriptional analysis in sorted cells. Experiments were conducted to validate liver neutrophil heterogeneity in Atp7b-/- mice. In vitro experiments were performed in ATP7B-knockout human hepatocellular carcinomas G2 cells and isolated bone marrow neutrophils to reveal the mechanism of neutrophil heterogeneity. RESULTS: Recruitment of neutrophils into the liver is observed in atp7b-/- zebrafish. Pharmacologic stimulation of neutrophils aggravates liver and behavior defects in atp7b-/- zebrafish. Transcriptional analysis in sorted liver neutrophils from atp7b-/- zebrafish reveals a distinct transcriptional profile characteristic of N2 neutrophils. Furthermore, liver N2 neutrophils also were observed in ATP7B-knockout mice, and pharmacologically targeted transforming growth factor ß1, DNA methyltransferase, or signal transducer and activator of transcription 3 reduces liver N2 neutrophils and improves liver function and alleviates liver inflammation and fibrosis in ATP7B-knockout mice. Epigenetic silencing of Socs3 expression by transforming growth factor ß1 contributes to N2-neutrophil polarization in isolated bone marrow neutrophils. CONCLUSIONS: Our findings provide a novel prospect that pharmacologic modulation of N2-neutrophil activity should be explored as an alternative therapeutic to improve liver function in Wilson's disease.

Activation of HIF-1 signaling ameliorates liver steatosis in zebrafish atp7b deficiency (Wilson's disease) models.

Wilson's disease is an autosomal recessive disease characterized by excess copper accumulated in the liver and brain. It is caused by mutations in the copper transporter gene ATP7B. However, based on the poor understanding of the transcriptional program involved in the pathogenesis of Wilson's disease and the lack of more safe and efficient therapies, the identification of novel pathways and the establishment of complementary model systems of Wilson's disease are urgently needed. Herein, we generated two zebrafish atp7b-mutant lines using the CRISPR/Cas9 editing system, and the mutants developed hepatic and behavioral deficits similar to those observed in humans with Wilson's disease. Interestingly, we found that atp7b-deficient zebrafish embryos developed liver steatosis under low-dose Cu exposure, and behavioral deficits appeared under high-dose Cu exposure. Analyses of publicly available transcriptomic data from ATP7B-knockout HepG2 cells demonstrated that the HIF-1 signaling pathway is downregulated in ATP7B-knockout HepG2 cells compared with wildtype cells following Cu exposure. The HIF-1 signaling pathway was also downregulated in our atp7b-deficient zebrafish mutants following Cu exposure. Furthermore, we demonstrate that activation of the HIF-1 signaling pathway with the chemical compound FG-4592 or DMOG ameliorates liver steatosis and reduces accumulated Cu levels in zebrafish atp7b deficiency models. These findings introduce a novel prospect that modulation of the HIF-1 signaling pathway should be explored as a novel strategy to reduce copper toxicity in Wilson's disease patients.

Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis.

Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn) efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC). However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn. Interestingly, slc30a10 mutants developed neurological deficits in adulthood, as well as environmental Mn-induced manganism in the embryonic stage; moreover, mutant animals had impaired dopaminergic and GABAergic signaling. Finally, mutant animals developed steatosis, liver fibrosis, and polycythemia accompanied by increased epo expression. This phenotype was rescued partially by EDTA- CaNa2 chelation therapy and iron supplementation. Interestingly, prior to the onset of slc30a10 expression, expressing ATP2C1 (ATPase secretory pathway Ca2+ transporting 1) protected mutant embryos from Mn exposure, suggesting a compensatory role for Atp2c1 in the absence of Slc30a10. Notably, expressing either wild-type or mutant forms of SLC30A10 was sufficient to inhibit the effect of ATP2C1 in response to Mn challenge in both zebrafish embryos and HeLa cells. These findings suggest that either activating ATP2C1 or restoring the Mn-induced trafficking of ATP2C1 can reduce Mn accumulation, providing a possible target for treating HMDPC.

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs.

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle.