Stimulation of Liver Fibrosis by N2 Neutrophils in Wilson’s DiseaseSummary

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 β1, 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 β1 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

Functional crosstalk between SLC30A10 and ATP2C1 in HeLa cells.

<p>(A) HeLa cells were immunostained for ATP2C1 (green) and the Mn sensor protein GPP130 (blue). Following overnight exposure to 300 μM Mn, most of the ATP2C1 was translocated to the Golgi apparatus, and GPP130 was degraded. (B) HeLa cells were transfected with SLC30A10-Q308Stop-flag (Q308Stop, a mutant form of SLC30A10 in which codon 308 is changed to a stop codon); the cells were then immunostained for flag (red) and GPP130 (blue). Following Mn exposure, only half of the GPP130 was degraded. (C) Quantitative analysis of GPP130 immunoreactivity. (D) HeLa cells were transfected with galactosyltransferase-RFP (GalT) plasmid to label the Golgi apparatus (red); the cells were then immunostained for ATP2C1 (green). Following Mn exposure, most of the ATP2C1 was translocated to the Golgi apparatus, where it co-localized with GalT. (E-G) HeLa cells were co-transfected with the indicated constructs (E: GalT-RFP and SLC30A10-FLAG; F: GalT-RFP and SLC30A10-Q308Stop-flag; G: GalT-RFP and SLC30A10-1st-2nd-exons-deletion-flag, a mutant form of SLC30A10 in which the first and second exons are deleted); the cells were then immunostained for ATP2C1 (green) and FLAG (blue). (H) HeLa cells were mock-transfected cells or transfected with the indicated construct. After overnight exposure to Mn (500 μM), Mn concentration was measure using IPC-MS. ***<i>p</i><0.001.</p

Schematic model depicting the proposed relationship between SLC30A10 and ATP2C1 in mediating Mn efflux.

<p>(Top) In the absence of SLC30A10, cells under Mn stress use ATP2C1 to clear Mn from the cytoplasm via Golgi vesicular trafficking. (Middle) When SLC30A10 levels are sufficient, cells under Mn stress use SLC30A10 as the primary Mn exporter, thereby bypassing the ATP2C1-mediated pathway. (Bottom) When cells express a mutant form of SLC30A10, Mn accumulates in the cytoplasm, causing Mn-induced toxicity; in addition, the ATP2C1-mediated pathway is reduced, limiting the efflux of Mn via Golgi vesicular trafficking.</p

<i>Slc30a10</i> mutant zebrafish develop polycythemia, and chelation treatments improves the phenotype in mutant embryos.

<p>(A) Summary of the relative numbers of erythrocytes in 3-week-old and 1-week-old embryos (n = 6 sets of 50 embryos/group). (B and C) <i>Epo</i> expression was measured in 3-week-old and 1-week-old mutant zebrafish (B) and in the liver of adult zebrafish (C); n = 3 sets of 20 adults/group. (D and E) Mn-induced locomotor defects (D) and the darker color in liver (E) were rescued by treating embryos with EDTA-CaNa₂ (chelator, n = 3 sets of 20 embryos/group). (F-G) Example images of a Mn-exposed mutant embryo, showing a darker colored liver (F), and a Mn-exposed mutant embryo following EDTA-CaNa₂ treatment (G). (H) Following exposure to 300 μM Mn, mutant embryos were transferred to either Holt buffer alone or Holt buffer containing EDTA-CaNa₂, and locomotion was measured (n = 3 sets of 20 embryos/group). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001.</p

<i>Slc30a10</i> mutants develop impaired neurological function.

<p>(A) Example images of affected mutant adults and their unaffected siblings; note the thinner body shape of the affected siblings. (B) Summary of body weight of affected adults and unaffected siblings (n = 4 adults/group). (C) Affected mutants have higher Mn levels compared to their unaffected siblings (n = 4 adults/group). (D) Example movement traces of affected adults and their unaffected siblings. (E) Affected adults swim shorter distances than their unaffected siblings (n = 6 adults/group). (F) Summary of the reproductive capacity of WT and mutant adults (n = 20 adults/group). (G) Example images of a wild-type and mutant embryo, showing a darker color in the brain and liver (red arrows) in the mutant. (H) Mn exposure causes a larger increase in Mn accumulation in mutant animals compared to wild-type animals (n = 3 sets of 200 embryos/group). (I) Example images of two mutant embryos after Mn exposure. Note the distorted body shape of the Mn-treated mutant. (J) Locomotion (measured as swimming and a normal escape response) is reduced in mutant embryos following Mn treatment, whereas wild-type animals are not affected (n = 3 sets of 20 embryos/group). (K) Mutant embryos exposed to Mn swim a shorter distance than untreated mutants and wild-type embryos (n = 6 embryos/group). (L) <i>Slc32a1</i> expression is reduced in both wild-type and <i>slc30a10</i> embryos following Mn exposure (n = 3 sets of 20 embryos/group). (M-N) <i>Gabb1a</i> (M) and <i>gabb1b</i> (N) expression is reduced in Mn-exposed <i>slc30a10</i> mutants (n = 3 sets of 20 embryos/group). (O) Western blot analysis of Gad65/67 in wild-type and mutant embryos treated with or without Mn. (P-Q) <i>In situ</i> hybridization of <i>dat</i> and <i>th</i> in mutant embryos following Mn exposure, showing reduced expression of both genes compared to untreated mutants and WT embryos. (R) TUNEL staining in the brain of mutant embryos following Mn exposure, showing increased apoptosis compared to untreated mutants and WT embryos. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001; in K‒O, groups with different letters differed significantly (<i>p</i><0.05).</p

<i>Slc30a10</i> mutant zebrafish develop liver damage.

<p>(A) Oil red O staining shows that mutant embryos have a slightly fatty liver (red arrows), which was worsened upon exposure to Mn. (B-C) HE staining (B) and Oil red O staining (C) of frozen sections showing hepatic steatosis in mutant embryos following Mn exposure. (D) Example images of a wild-type and mutant embryo under Mn exposure, showing a darker colored liver in the mutant (red arrow). (E) Dose-response curve showing the percentage of embryos with liver color change versus Mn concentration (n = 3 sets of 20 embryos/group). (F) Image of the liver of a wild-type and mutant adult, showing severe fibrosis in the mutant liver with sirius red staining. (G-H) The fibrosis markers <i>col1a1a</i> and <i>ctgfa</i> were measured in the liver of both male and female WT and mutant animals (n = 3 sets of 20 adults/group; *<i>p</i><0.05 and **<i>p</i><0.01).</p

Slc30a10 functions as a Mn exporter in zebrafish.

<p>(A) <i>In situ</i> hybridization of wild-type zebrafish using the antisense <i>slc30a10</i> probe, showing expression in the brain and liver (red arrows), as well as the YSL (red arrowheads). A sense probe was used as a negative control. (B) Semi-quantitative RT-PCR of <i>slc30a10</i> mRNA in wild-type 1–7 dpf embryos, 14 dpf larvae, and 21 dpf larvae, showing the onset of expression at 5 dpf. (C) DNA and corresponding amino acid sequences of the wild-type (WT), 10-bp deletion (-10), and 1-bp insertion (+1) <i>slc30a10</i> alleles following CRISPR/Cas9-based editing. (D) Summary of <i>slc30a10</i> expression in WT and both <i>slc30a10</i> mutant lines (n = 3 sets of 50 embryos/group). (E-G) Mn, Fe, and Zn concentration was measured in wild-type and mutant 1-week-old embryos (E; n = 6 sets of 1000 embryos/group), 3-week-old larvae (F; n = 3 sets of 50 larvae/group), and 4-month-old adults (G; n = 3 adults/group). (H-J) Heterozygous embryos were exposed to the indicated concentrations of Mn²⁺ (H), Fe³⁺ (ferric ammonium citrate, FAC; I), or Zn²⁺ (J) for 24 hours at 5 dpf. At 6 dpf, <i>slc30a10</i> expression was measured; n = 3 sets of embryos/group. *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001; N.S., not significant (<i>p</i>>0.05).</p