Using two different genetic mouse knockouts of β-catenin, one with β-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived β-catenin-positive hepatocytes and resolution of injury. KO1 showed persistent loss of β-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of β-catenin, NFκB, and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or β-catenin led to NF-κB activation, DR, and inflammation. Thus, we report a novel β-catenin-NFκB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis,  The liver has an incredible capacity to repair itself or ‘regenerate’ – that is, it has the ability to replace damaged tissue with new tissue. In order to do this, the organ relies on hepatocytes (the cells that form the liver) and bile duct cells (the cells that form the biliary ducts) dividing and transforming into each other to repair and replace damaged tissue, in case the insult is dire.During long-lasting or chronic liver injury, bile duct cells undergo a process called ‘ductular reaction’, which causes the cells to multiply and produce proteins that stimulate inflammation, and can lead to liver scarring (fibrosis). Ductular reaction is a hallmark of severe liver disease, and different diseases exhibit ductular reactions with distinct features. For example, in cystic fibrosis, a unique type of ductular reaction occurs at late stages, accompanied by both inflammation and fibrosis. Despite the role that ductular reaction plays in liver disease, it is not well understood how it works at the molecular level.Hu et al. set out to investigate how a protein called β-catenin – which can cause many types of cells to proliferate – is involved in ductular reaction. They used three types of mice for their experiments: wild-type mice, which were not genetically modified; and two strains of genetically modified mice. One of these mutant mice did not produce β-catenin in biliary duct cells, while the other lacked β-catenin both in biliary duct cells and in hepatocytes.After a short liver injury – which Hu et al. caused by feeding the mice a specific diet – the wild-type mice were able to regenerate and repair the liver without exhibiting any ductular reaction. The mutant mice that lacked β-catenin in hepatocytes showed a temporary ductular reaction, and ultimately repaired their livers by turning bile duct cells into hepatocytes. On the other hand, the mutant mice lacking β-catenin in both hepatocytes and bile duct cells displayed sustained ductular reactions, inflammation and fibrosis, which looked like that seen in patients with liver disease associated to cystic fibrosis. Further probing showed that β-catenin interacts with a protein called CTFR, which is involved in cystic fibrosis. When bile duct cells lack either of these proteins, another protein called NF-B gets activated, which causes the ductular reaction, leading to inflammation and fibrosis.The findings of Hu et al. shed light on the role of β-catenin in ductular reaction. Further, the results show a previously unknown interaction between β-catenin, CTFR and NF-B, which could lead to better treatments for cystic fibrosis in the future. The liver possesses unique regenerative potential. During chronic liver injury, however, liver fibrosis accompanies regeneration and can progress to cirrhosis, which can then progress to end-stage liver disease (ESLD) or hepatocellular cancer (HCC) . CurrentAs an important hepithelial cell type, BECs are known to undergo proliferation to replace dying BECs in cholangiopathies or cystic liver diseases, as well as can under phenotypic switch to generate de novo hepatocytes when hepatocytes are chronically injured and/or are unable to optimally proliferate, phenomena termed as ductular reaction (DR) . ReactivCre recombinase under a hepatocyte-specific thyroxine-binding globulin (TBG) promoter (AAV8-TBG-Cre) into the β-catenin-floxed mice  β-catenin knockout (KO1) mice generated by breeding β-catenin-floxed and albumin-cre mice show delayed liver regeneration (LR) after partial hepatectomy or after toxicant-induced liver injury . When KOxed mice . Intriguxed mice .In the current study, we investigate hepatic injury and repair in KO2 and KO1 mice challenged for 2 weeks with CDE diet and allowed to recover on normal diet for 2 weeks, 3 months, and 6 months. Intriguingly, we observed highly divergent injury-repair responses in the two models. KO2 mice showed progressive repair through expansion of BEC-derived β-catenin-positive hepatocytes and resolution of inflammation, DR, and fibrosis. However, KO1 display progressive and peculiar DR composed of numerous small luminal structures lined by a single layer of BECs, even after being on normal diet for 6 months, which is associated with fibrosis and inflammation, and is reminiscent of cystic fibrosis (CF)-like morphology. We identify a unique interactome of β-catenin, p65 subunit of NF-κB and cystic fibrosis transmembranous conductance regulator (CFTR) in BECs and show perturbations in these interactions, leading to excessive NF-κB activation and inflammation in BECs in both KO1 and CF patients.flox/flox; Rosa-stopflox/flox-EYFPCtnnb1 mice, as compared to WT2 mice, generated by injecting AAV8-TBG-Cre into +/+; Rosa-stopflox/flox-EYFPCtnnb1 mice , generated by delivering AAV8-TBG-Cre into YFP mice . And thaYFP mice . To specYFP mice . KO2 micYFP mice . AlkalinYFP mice .Ctnnb1) expression showed increasing expression in KO2 livers over time, becoming comparable to WT2 at 6 months , a well-known inhibitor of matrix metalloproteinases, known for a role in extracellular matrix degradation, was determined next as a possible mechanism of fibrosis resolution  mice lacking β-catenin in hepatocytes and BECs, and their wild-type littermates (WT1) on CDE diet for 2 weeks and allowed recovery on normal diet for 2 weeks, 3 months, and 6 months  . SignifiTimp1 correlated with reduced fibrosis in KO2 at 6 months of recovery, we next investigated its levels in KO1 and WT1. Timp1 tended to be upregulated in KO1 mice at all times but significantly at 6-month recovery time  integrity and could explain differences in DR and fibrosis between KO2 and KO1 mice. Temporal disruption of cell-cell junctions has been associated with pathologies in hepatobiliary injury including after CDE diet . HoweverPreviously, the presence of immune cell infiltration has been shown to be essential in the development of DR and fibrosis. Mice lacking Th1 immune signaling or interferon-γ showed impaired DR and decreased fibrosis after CDE diet . To addrTo determine if these inflammatory cells were close to DR, we performed triple immunofluorescence (IF) for PanCK, CD45, and myofibroblast marker α-smooth muscle actin (αSMA) . At 2-weAdgre1, gene encoding macrophage marker F4/80, showed significant increase in KO1 compared to WT1 mice at 3-month recovery (Itgam (CD11b), and infiltrate liver during injury, express pro-inflammatory cytokines, and are involved in both progression and recovery phases of fibrosis  or tumor necrosis factor-α challenge . FurtherTo verify if nuclear p65 indicated NF-κB activation, 84 downstream target genes were checked by RT-PCR array . Relative to WT1, we found a striking upregulation in the expression of 44% of genes (37/84) in KO1, whereas 92% of genes (77/84) in KO2 livers were unchanged at 6 months of recovery . ClusterAltogether, these data suggest a pronounced and prolonged NF-κB activation in BECs lacking β-catenin along with periportal inflammation during recovery phase from CDE injury, whereas presence of β-catenin dampened NF-κB activation in BECs to curbed inflammation and assist in recovery from the same injury.To more conclusively address the relationship between β-catenin and NF-κB in BECs directly, we utilized immortalized mouse small cholangiocyte cells (SMCCs), which were transfected with control- or β-catenin siRNA together with either β-catenin-TCF TOPFlash reporter or p65 luciferase reporter. Knockdown of β-catenin in SMCCs, shown by a significant decrease in TOPFlash activity, induced p65 luciferase activity, which was three times greater than caused by 100 ng/ml LPS stimulation . ConversTo address how β-catenin regulates NF-κB activity, SMCCs transfected with control- or Ctnnb1-siRNA were cultured with or without LPS, and subjected to cell fractionation to isolate nuclear- and cytoplasmic-enriched fractions. By WB, at baseline, notably higher levels of both p65 and β-catenin were evident in the cytoplasmic and not nuclear fraction. Upon β-catenin silencing, significantly higher levels of p65 were found in the nuclear compartment versus the controls, both without LPS, but more so following LPS treatment .Next, we modulated β-catenin activity in SMCCs to determine changes in global gene expression. Bulk RNA-seq was performed on β-catenin-silenced or control SMCCs, and on SMCCs transfected with S45Y-β-catenin or eGFP. Using a cutoff p-value ≤ 0.05 and abs(log2FC) ≥ 1.5, we found 335 β-catenin-regulated genes in BECs. Specifically, β-catenin-silenced SMCCs showed 75 upregulated and 122 downregulated, and β-catenin-active SMCCs showed 76 upregulated and 69 downregulated genes . While tNext, we investigated if β-catenin interacts with p65 in BECs. Whole-cell lysates from SMCCs and normal mouse liver were used to pull down p65. We identified robust β-catenin association with p65 in SMCCs . A faintAltogether, biochemical and IF studies identify a heretofore undescribed β-catenin-p65 complex in the cytoplasm of BECs. Further, β-catenin seems to prevent p65 nuclear translocation and NF-κB activation, and may be important in shutting off NF-κB activation when its signaling is no longer required. Absence of β-catenin in BECs enhances nuclear translocation of p65 at baseline, and more so in the presence of known positive regulators of NF-κB signaling such as LPS, and contributes to enhanced expression of genes involved in proliferation, inflammation, and fibrosis .Since persistent DR observed in KO1 at 3 months and 6 months after recovery from CDE injury showed unique morphology consisting of numerous small luminal structures lined by a single layer of PanCK-positive cells, we decided to interrogate livers from clinical cases exhibiting DR including alcoholic hepatitis (AH), polycystic liver disease (PLD), and CF. As seen by panCK IF, AH cases displayed DR with variable morphology including areas of luminal small DR (shown in representative case), whereas PLD showed large cysts lined by flattened BECs . DR in CCFTR gene. Since β-catenin-p65 interactions were observed in SMCCs, we next assessed if CFTR is interacting with this complex. We observed concomitant pulldown of both B-band (faster migrating core glycosylated immature form) as well as slowly migrating C-band (complex glycosylated form)  mutations in ed form) , along wed form) . To mimiactivity . Likewisnockdown . To quertivation .To query if β-catenin loss in BECs had any impact on CFTR levels, we examined total levels of CFTR in KO1 and WT1 at baseline and at 6 months of recovery from CDE diet. We did not observe any differences in the levels of total CFTR protein, suggesting that CFTR may be an upstream effector of β-catenin while β-catenin does not regulate CFTR levels in BECs .Thus, we identify important interactions between β-catenin, p65, and CFTR in BECs, and LOF of CFTR leads to destabilization of β-catenin, which in turn allows p65/NF-κB activation more readily in response to the available cues including the presence of cytokines. Also, β-catenin stabilization in BECs could tamper such enhanced p65 activation and may have potential therapeutic benefit in controlling unchecked NF-κB activation, inflammation, and fibrosis in reactive ductular cells.DR is a common hallmark of many chronic liver pathologies, although its morphology is heterogeneous ranging from isolated invasive ductular cells, luminal phenotype, and sometimes purely cystic . The sigCcl2 and Cxcl5  of adeno-associated virus serotype 8 encoding Cre recombinase under the hepatocyte-specific thyroid binding globulin promoter  followed by a 12-day washout period. The same protocol was utilized on +/+;Rosa-stopflox/flox-EYFPCtnnb1 mice to generate WT2 mice. When mice were 4–6 weeks old, choline-deficient diet (Envigo Teklad Diets) supplemented with 0.15% ethionine drinking water  was administered for 2 weeks. For recovery time points, animals were switched back to normal chow diet for up to 6 months. Serum biochemistry analysis was performed by automated methods at the University of Pittsburgh Medical Center clinical chemistry laboratory. All studies were performed according to the guidelines of the National Institutes of Health and the University of Pittsburgh Institutional Animal Use and Care Committee.All animals are housed in temperature and light-controlled facilities and are maintained in accordance with the Guide for Care and Use of Laboratory Animals and the Animal Welfare Act. Generation of eviously . Generateviously . In brieAll patient tissue sections were provided by the Pittsburgh Liver Research Center’s (PLRC’s) Clinical Biospecimen Repository and Processing Core (CBPRC), supported by P30DK120531. Sections from 5 patients with healthy liver, 12 patients with DR from AH (n = 10) and/or NASH (n = 2), 5 patients with DR associated with PLD, and 6 patients with DR in CF cases were triple stained with CK19, p65, and β-catenin for further analysis. Patient information from these groups of cases is listed in 2O2 for 10 min to quench endogenous peroxide activity. After washing with PBS, slides were blocked with Super Block  for 10 min or 10% goat serum in PBS for 10 min (p21). The primary antibodies were incubated at the following concentrations in antibody diluent: PBS + 1% BSA  with 0.1% Tween 20 : PanCK , cleaved caspase 3 , p-Erk1/2 , F4/80  for 1 hr at room temperature or at 4°C overnight: β-catenin  and p21 . Samples were washed with PBS three times and incubated with the appropriate biotinylated secondary antibody (Vector Laboratories) diluted 1:500 in antibody diluent for 30 min at room temperature. Samples were washed with PBS three times and sensitized with the Vectastain ABC kit  for 30 min. Following three washes with PBS, color was developed with DAB Peroxidase Substrate Kit , followed by quenching in distilled water for 5 min. Slides were counterstained with hematoxylin , dehydrated to xylene, and coverslips applied with Cytoseal XYL . For Sirius Red staining, samples were deparaffinized and incubated for 1 hr in Picro-Sirius Red Stain , washed twice in 0.5% acetic acid water, dehydrated to xylene, and coverslipped. Images were taken on a Zeiss Axioskop 40 inverted brightfield microscope.The IHC protocols have been described previously . In brieLiver tissue was fixed in 10% buffered formalin overnight, cryopreserved with 30% sucrose in PBS overnight, frozen in OCT compound , and stored at –80°C. OCT-embedded samples were cut into 5-µm sections, allowed to air-dry, and then washed in PBS. Antigen retrieval was performed through microwaving in pH 6 sodium citrate buffer. Slides were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature. Samples were washed three times with PBS and then blocked with 2% donkey serum in 0.1% Tween 20 in PBS (antibody diluent) for 30 min at room temperature. Antibodies were diluted as follows: PanCK , PCNA , p65 , β-catenin , CK-19  in antibody diluent, and incubated at 4°C overnight. Ly6G (clone: RB6-8C5) antibody was purchased from Thermo Fisher Scientific, Waltham, MA. Samples were washed three times in PBS and incubated with the proper fluorescent secondary antibody  diluted 1:400 in antibody diluent for 2 hr at room temperature. The αSMA antibody is directly conjugated to Cy3 and requires no secondary antibody. Samples were washed three times with PBS and incubated with DAPI  for 1 min. Samples were washed three times with PBS and mounted with fluoromount (SouthernBiotech) or ProLong Gold antifade reagent . Images were taken on a Nikon Eclipse Ti epifluorescence microscope or a Zeiss LSM700 confocal microscope.To determine BEC proliferation, for each sample seven images at 200× magnification of periportal regions were taken, and in each image the number of PanCK+/PCNA+ cells was manually counted. For quantification of Sirius Red, PanCK, CD45 staining, staining intensity was measured in ImageJ for 3–5 images at 100× magnification per mice sample. Positive area to whole area ratio was calculated as percent-positive area using the NIH ImageJ software. To determine p65 nuclear-positive cholangiocytes, four images from each animal or patient at 200× magnification of DR regions were counted.Gapdh and Rn18s), and fold-change was calculated utilizing the ΔΔ-Ct method. For RT-PCR arrays, 2 μl of cDNA, 7.5 μl of Power SYBR Green PCR Master Mix, and 4.5 μl of nuclease-free water were premixed and added to each well of RT² Profiler PCR Array Mouse NFκB Signaling Pathway  for target gene qPCR. RT-PCR arrays data were analyzed at GeneGlobe . The average of three housekeeping genes  was used for normalization. Volcano plot and clustergram were generated by the data analysis web portal mentioned above.Whole liver was homogenized in TRIzol  and nucleic acid was isolated through phenol-chloroform extraction. Cellular DNA was digested with DNA-free Kit , and RNA was reverse-transcribed into cDNA using SuperScript III . Real-time PCR was performed in technical triplicate on a StepOnePlus Real-Time PCR System  or on a Bio-Rad CFX96 Real-Time System using the Power SYBR Green PCR Master Mix . Target gene expression was normalized to the average of two housekeeping genes . The concentration of the protein was determined by the bicinchoninic acid assay. For IP, 1 mg of SMCC lysate was precleared with 40 μl of Protein A/G PLUS-Agarose  for 2 hr at 4°C. After centrifugation , the supernatant was incubated with 2 μg of p65 antibody , 2 μg of β-catenin antibody , or control IgG overnight at 4°C. The next day, samples were incubated with 40 μl of Protein A/G PLUS-Agarose for 1 hr at 4°C. The pellet was collected, washed with RIPA buffer for three times, resuspended in 10 μl of loading buffer, and subjected to electrophoresis. Protein lysate was separated on pre-cast 7.5% or 4–20% polyacrylamide gels (Bio-Rad) and transferred to the PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 75 min with 5% skim milk  or 5% BSA in Blotto buffer , and incubated with primary antibodies at 4°C overnight at the following concentrations: β-catenin , p65 , CFTR , GAPDH , and histone H3 . Membranes were washed in Blotto buffer and incubated with the appropriate HRP-conjugated secondary antibody for 75 min at room temperature. Membranes were washed with Blotto buffer, and bands were developed utilizing SuperSignal West Pico Chemiluminescent Substrate  and visualized by autoradiography.2 maintained at 37°C. Cells were mycoplasma negative tested with MycoAlert Mycoplasma Detection Kit . For p65 reporter assay, cells were transfected with 2 μg of p65 reporter and 0.2 μg of Renilla  together with 2 μg of either eGFP (control) or S45Y to overexpress constitutively active β-catenin using Lipofectamine 3000 Transfection Reagent , or together with si-Control  and si-β-catenin  to knock down β-catenin using Lipofectamine RNAiMAX Transfection Reagent . For TOPFlash reporter assay, p65 reporter above was replaced by TOPFlash plasmid. Cells were treated with 100 ng/ml of LPS 6 hr before harvest. si-CFTR  was used to knock down CFTR in SMCCs. Cells were harvested at 48 hr, and luciferase signals were got using Dual-Luciferase Reporter Assay System  and normalized to the value of Renilla.SMCCs , and human CCA cell lines MzChA and HuCCT1  were seeded on six-well plates in a humidity-saturated incubator with 5% COTotal hepatic bile acids were measured using the Mouse Total Bile Acids Assay Kit from Crystal Chem , as per the manufacturer’s instructions. To isolate total bile acids from liver, 50–100 mg frozen liver tissue was homogenized in 70% ethanol at room temperature, then samples were incubated in capped glass tubes at 50°C for 2 hr. The homogenates were centrifuged at 6000 g for 10 min to collect the supernatant. Total bile acid concentrations were determined using the calibration curve from the standard provided in the kit and the mean change in absorbance value for each sample.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE155981).Twelve SMCC RNA samples were measured: three for CTNNB1 activation (SMCC-S45Y), three for CTNNB1 activation control (SMCC-eGFP), three for CTNNB1 silencing (SMCC-si-β-catenin), and three for CTNNB1 silencing control (SMCC-si-Control). In total, 12 RNA-seq libraries were sequenced. For each library, quality control was performed to each raw sequencing data by tool FastQC. Based on the QC results, low-quality reads and adapter sequences were filtered out by tool Trimmomatic . SurviviFor analysis of serum biochemistry between two groups, a two-tailed t-test was performed. For analysis of cell counts, such as proliferating BECs, a Mann–Whitney U test was performed. A p<0.05 was considered significant, and plots are mean ± SD. Detailed statistic information for each assay is given in the figure legends. All statistical analysis and graph generation were performed using GraphPad Prism software. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study explores the roles of biliary epithelial cells and hepatocytes in repair of liver injury. They make the interesting observation that the loss of β-Catenin in both binary epithelial cells and hepatocytes results in persistent inflammation and fibrosis with persistent activation of NFκB, wheres the loss in just hepatocytes results in healing. This pathology was reminiscent of liver damage seen in cystic fibrosis leading the group to identify an interaction with CFTR. These studies will encourage others to explore the relative contributions of biliary epithelial cells and hepatocytes in liver injury and repair.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Paul Noble as the Senior Editor and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Xin Chen (Reviewer #1).Thank you for submitting your article "β-Catenin-NFκB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. The IHC images of β-Catenin in Figures 1C and 3C are very hard to interpret. A higher resolution images or inset images will be highly helpful.2. Also, what happens to GS IHC in the two mouse Ctnnb1 KO model?3. It would be helpful to quantify CD45(+) cells in Figures 5A and 5B.4. For Figure7, does bCatenin and P65 interaction occur in cytoplasm or nuclear in BECs? Please clarify.5. Sup. 1A showing that PanCK(+) cells are proliferating is very important. However, the staining is not entirely convincing (maybe due to the low resolution) that those PCNA(+) cells are also PanCK(+) (seem to be the adjacent cells are positive for PCNA). Perhaps better images or additional DR markers will be helpful.6. In the introduction, the author mentioned that YAP has been implicated in DR. What happens to YAP in these Ctnnb1 KO liver samples? Some studies or discussion would be helpful.7. Most of the experimental groups for KO mice analyzed only n=3 or 4, which caused a big variation and reduced statistical power. This point should be improved throughout the study.8. Figure 2A,B examined fibrosis. Sirius red staining showed fibrosis is similar degrees in WT2 and KO2 mice 2 weeks after recovery and more fibrosis in KO2 3 months after recovery. The pictures are inconsistent with the quantification shown in Figure 2B.9. Figure 2C examined the expression of fibrogenic genes, Col1a1 and TGFb2. However, these data did not support the statement in the text because of not statistically different. What are other collagen and TGF-b genes?10. Figure 2D showed DR in WT2 and KO2 during the recovery. DR is also associated with liver regeneration. What is the hepatocyte proliferation here? IHC for DR should also be quantified.11. Figure 2F showed Timp1 expression for understanding matrix degradation. What is MMP expression?12. In Figure 4A,B, almost no fibrosis is observed in WT1, which is inconsistent with WT2 in Figure 2A. WT1 and WT2 should theoretically show similar degrees of fibrosis. Sirius red staining showed a significant reduction of fibrosis in KO1 after 2 weeks of recovery. However, the quantification  did not show that. Also, Figure 4C collagen 2C data did not support the authors' concept.13. In Figure 6, the authors assessed NF-κBp65 nuclear translocation mainly in CK19 positive cells. Also, in the text, the authors mentioned "NF-κB, the master regulator of immune cell response." Is NF-κB activation in CK19 positive cells related to immune cell infiltration that the study showed in Figure 5. If so, how? There is a gap between immune cell infiltration and NF-κB activation in BECs.14. Figure 8 showed the relationship between b-catenin, NF-κBp65, and CFTR. This figure suggested CF livers had decreased b-catenin expression that enhances NF-κB activity, which induces the expression of NF-κB target genes. The authors suggested CF livers may have a similar phenotype caused in KO1 mice. Do KO1 livers have altered CFTR expression or activity? If the study suggests the interaction of b-catenin and NF-κB with CFTR, KO1 could have altered CFTR expression and some phenotypes related to CFTR inhibition.15. The authors should also provide histologic examination of liver injury in mice by HandE staining.16. To study the repair of liver damage in KO1 and KO2 mice, it is necessary to determine whether KO1 and KO2 mice will spontaneously cause liver damage. Therefore, it is important to provide groups of KO1 and KO2 mice without CDE diet.17. There seems to be a significant fat accumulation in KO1 and KO2 mice compared to WTs mice on CDE diet for 2w in liver tissue section. Therefore, the author should also assess fat metabolism.18. Line 176, "KO2" should be "KO1".Reviewer #1 (Recommendations for the authors):In this manuscript, Hu et al., analyzed DR in hepatocyte specific Ctnnb1 KO or BEC/hepatocyte specific Ctnnb1 KO mice. The studies identified a novel biochemical interaction between β-catenin, NF-κB and CTFR in BECs, which are implicated in DR induced inflammation and fibrosis.Reviewer #2 (Recommendations for the authors):The major strengths of the paper are the use of two distinct Ctnnb1 KO mouse models for comparison, with no obvious method/result weaknesses. The likely impact of the studies on liver regeneration is quite high. Overall this is an excellent study of interest to scientists who are interested in liver regeneration.Hu et al., investigated the role of b-catenin in BECs in the regulation of NF-κB activity and the recovery from CDE-induced chronic liver injury. Also, the study suggests b-catenin and NF-κB activation may have significant roles in CF livers. First, the study compared the recovery patterns from CDE-induced chronic liver injury in mice deficient in b-catenin in hepithelial cells and in hepatocytes only. The study found b-catenin activity is crucial for the recovery of chronic liver inflammation, fibrosis, and DR. The authors found reduced activity of b-catenin is associated with prolonged NF-κB activation in BECs. The authors also found that CF patient livers had reduced b-catenin expression and NF-κB activity, and that b-catenin, NF-κB, and CFTR form the complex and regulate NF-κB activity in BECs. Overall, the study should be of interest to the researchers who investigate liver regeneration, its relation to BEC biology, and cystic fibrosis.Strengths:1. The use of two different knockout mice; one is hepithelial cell-specific b-catenin knockout mice and the other is hepatocyte only-specific b-catenin knockout mice.2. Understanding the recovery phase of chronic liver injury is important.3. The crosstalk between b-catenin and NF-κB in BECs is of interest.4. The interaction of b-catenin and NF-κB with CFTR is clinically relevant.Weaknesses:1. Most of the experimental groups for KO mice analyzed only n=3 or 4, which caused a big variation and reduced statistical power. This point should be improved throughout the study.2. Figure 8 showed the relationship between b-catenin, NF-κBp65, and CFTR. This figure suggested CF livers had decreased b-catenin expression that enhances NF-κB activity, which induces the expression of NF-κB target genes. The authors suggested CF livers may have a similar phenotype caused in KO1 mice. However, it is unclear whether KO1 livers have altered CFTR expression or activity. If the study suggests the interaction of b-catenin and NF-κB with CFTR, KO1 could have altered CFTR expression and some phenotypes related to CFTR inhibition.Reviewer #3 (Recommendations for the authors):In this study, the authors studied hepatic injury and repair in two different genetic knockouts of β-catenin, one with β-catenin loss is hepatocytes and biliary epithelial cells (KO1), and another with loss in only hepatocytes (KO2) mice. These mice were challenged for 2w with choline-deficient, ethionine-supplemented (CDE) diet and allowed to recover on normal diet for 2w, 3m and 6m. KO2 show gradual liver repopulation with biliary epithelial cell-derived β-catenin-positive hepatocytes, and resolution of injury. KO1 showed persistent loss of β-catenin, NF-κB activation in biliary epithelial cells, progressive ductular reaction and fibrosis. They identified interactions of β-catenin, NF-κB and CFTR in biliary epithelial cells. Loss of CFTR or β-catenin led to NF-κB activation, ductular reaction and inflammation. Although data provided here by the authors could have interesting implications, there are several concerns about the research design.Specific points:1. The authors should also provide histologic examination of liver injury in mice by HandE staining.2. To study the repair of liver damage in KO1 and KO2 mice, it is necessary to determine whether KO1 and KO2 mice will spontaneously cause liver damage. Therefore, it is important to provide groups of KO1 and KO2 mice without CDE diet.3. There seems to be a significant fat accumulation in KO1 and KO2 mice compared to WTs mice on CDE diet for 2w in liver tissue section. Therefore, the authors should also assess fat metabolism. Essential revisions:1. The IHC images of β-Catenin in Figures 1C and 3C are very hard to interpret. A higher resolution images or inset images will be highly helpful.We have now updated figures 1C and 3C with better quality images for β-catenin immunohistochemistry. We have included 100x images here. We have also added higher magnification images (200x) as supplemental figure for both KO1 and KO2 models for β-catenin staining .2. Also, what happens to GS IHC in the two mouse Ctnnb1 KO model?That is an excellent point. We have added IHC for GS for both KO1 and KO2 livers at 3 and 6 months of recovery from 2-week CDE diet. We observed continued absence of GS in KO1 while GS progressively re-appears in zone-3 hepatocytes in KO2. This data is presented as a supplemental figure S2.3. It would be helpful to quantify CD45(+) cells in Figures 5A and 5B.This has now been included in Supplemental Figure 5 and shows statistically significantly higher inflammation in KO1 at 6 months as compared to all other time points although KO1 are always tending to have higher CD45 cells than WT1 at comparable time-points. This is in contrast to KO2 which progressively and significantly resolved CD45 infiltrate at 3 months onwards after recovery from CDE.4. For Figure7, does bCatenin and P65 interaction occur in cytoplasm or nuclear in BECs? Please clarify.Based on colocalization studies using immunofluorescence (now supplementary figure 9F), most of the interaction of β-catenin and p65 occurs in the cytoplasm of the BECs in vivo. in vitro data in SMCCc shows that most β-catenin and p65 are located in cytoplasmic compartment . Upon β-catenin knockdown, there is a modest increase in nuclear p65 levels and activity, which is more pronounced following LPS treatment. This data also supports that β-catenin-p65 binding occurs in cytoplasm at baseline which prevents nuclear translocation of p65 spontaneously or after treatment with LPS. We have better clarified this in both results and discussion5. Sup. 1A showing that PanCK(+) cells are proliferating is very important. However, the staining is not entirely convincing (maybe due to the low resolution) that those PCNA(+) cells are also PanCK(+) (seem to be the adjacent cells are positive for PCNA). Perhaps better images or additional DR markers will be helpful.+ cells to be present in ducts (PanCK+) as well as in nonductal (PanCK-) cells in the periportal area. The white arrowhead identifies only PanCK+; PCNA+ cells, which were carefully identified and quantified. We wanted to show a low magnification image to capture a larger representative area but we do quantify these images to present as a bar graph which is accurate.Supplementary Figure 1A  (right panel) shows PCNA6. In the introduction, the author mentioned that YAP has been implicated in DR. What happens to YAP in these Ctnnb1 KO liver samples? Some studies or discussion would be helpful.YAP1 is seen mostly in biliary epithelial cells in an adult liver and contributes to their survival and proliferation. Its deletion from cholangiocytes disrupts homeostasis leading to bile duct paucity and cholestatic injury. Since there is increased DR in KO1 at 3 and 6 months of recovery from CDE, YAP1 levels are expected to increase simply due to their expression in these cells. Indeed we see increased YAP1  in DR in7. Most of the experimental groups for KO mice analyzed only n=3 or 4, which caused a big variation and reduced statistical power. This point should be improved throughout the study.We agree that our study uses n≥3 mice for control and KO groups and at various time points. This is chiefly due to allocation of the minimal numbers of mice to multiple time points for analysis that still allow us to perform statistics and allow some generalizable conclusions. During COVID-19 pandemic, there were additional restrictions placed on how many mice we were allowed to keep. Additionally, we believe that the variability seen in RT-PCR analysis is most likely due to diversity in cell composition of randomly selected liver pieces that could have varying degree of ductular reaction etc. We have been careful to not over-interpret results. We have been transparent about all significant and insignificant differences in various markers and mentioned trends versus significance, where applicable.8. Figure 2A,B examined fibrosis. Sirius red staining showed fibrosis is similar degrees in WT2 and KO2 mice 2 weeks after recovery and more fibrosis in KO2 3 months after recovery. The pictures are inconsistent with the quantification shown in Figure 2B.We apologize for this confusion. While we were careful to include the representative images, we have now replaced these images with better quality 100x images. We do observe a clear difference in Sirius Red staining (Red color) in KO2 as compared to WT2 at the time-points indicated as was assessed by densitometry for Sirius red staining. All statistics were done using one way ANOVA.9. Figure 2C examined the expression of fibrogenic genes, Col1a1 and TGFb2. However, these data did not support the statement in the text because of not statistically different. What are other collagen and TGF-b genes?Again, we are confident that the results are accurate and while the RT-PCR does not show significant difference in gene expression of these various genes indicative of ongoing fibrosis, the trends are clear. Eventually though, at 6 months, our key time point of concern, the fibrosis has subsided in KO2, and comparable to WT2. The possible reason for the variability in expression of genes within biological replicates is likely due to diversity in cell composition of randomly selected liver pieces for RT-PCR. One way ANOVA, which is more stringent than unpaired T test was used for significance.10. Figure 2D showed DR in WT2 and KO2 during the recovery. DR is also associated with liver regeneration. What is the hepatocyte proliferation here? IHC for DR should also be quantified.We have previously published hepatocyte proliferation in this model (PMID: 30215850). There is decrease in hepatocyte proliferation in KO2 following CDE injury and recovery due to lack of β-catenin, but as cholangiocyte-derived hepatocytes appear, they show more profound proliferation and gradually repopulate the KO2 liver. We have quantified IHC for panCK for DR for both figure 2D and 4D and data is presented as supplemental figure S3.11. Figure 2F showed Timp1 expression for understanding matrix degradation. What is MMP expression?While this is a good point, as the reviewers are aware, there are multiple MMPs and can be secreted my multiple cell types. Hence a comprehensive analysis of all MMPs might not be feasible. We assessed MMP9 and MMP13 on the available whole liver RNA and data is presented in  . As can12. In Figure 4A,B, almost no fibrosis is observed in WT1, which is inconsistent with WT2 in Figure 2A. WT1 and WT2 should theoretically show similar degrees of fibrosis. Sirius red staining showed a significant reduction of fibrosis in KO1 after 2 weeks of recovery. However, the quantification  did not show that. Also, Figure 4C collagen 2C data did not support the authors' concept.We apologize for these errors and appreciate the reviewer’s astute comments. The images in figure 4A for WT1 have now been replaced by more representative images. The reviewer is also correct and WT1 and WT2 showed comparable but low fibrosis after CDE injury. The KO1 also continue to show higher fibrosis than controls at all time points and do not show any decrease in fibrosis at 2 weeks of recovery. A better image has been included and text has been corrected. The Sirius red quantification in figure 4B is indeed correct. However, the collagen data, like mentioned previously, shows higher trends of expression which did not achieved significance as assessed by one way ANOVA. This is likely due to variability of DR observed in pieces of tissue used for RNA isolation and RT-PCR.13. In Figure 6, the authors assessed NF-κBp65 nuclear translocation mainly in CK19 positive cells. Also, in the text, the authors mentioned "NF-κB, the master regulator of immune cell response." Is NF-κB activation in CK19 positive cells related to immune cell infiltration that the study showed in Figure 5. If so, how? There is a gap between immune cell infiltration and NF-κB activation in BECs.CCl2 and Cxcl5 that are known drivers of inflammation. Simultaneously we see increased CD45 positive cells in KO1 in periportal region where p65 is observed in reactive cholangiocytes. KO2 have no inflammation and no nuclear p65 in cholangiocytes. Taken together, NF-κB in cholangiocytes through expression of chemokines, is likely the driver of inflammation. We apologize for not making this clear but have now added clarity to this end in the Discussion section.This is a valid point. We show using in vivo data first that absence of β-catenin in BECs led to increased nuclear p65 which was associated with increase expression of chemokines . However, since whole livers were used, we wanted to verify if this was also occurring in isolated BECs. For this we used SM-CC cells and upon β-catenin knockdown, showed increased p65 reporter activity along with increased nuclear p65. There was simultaneous increase in expression of similar chemokines like 14. Figure 8 showed the relationship between b-catenin, NF-κBp65, and CFTR. This figure suggested CF livers had decreased b-catenin expression that enhances NF-κB activity, which induces the expression of NF-κB target genes. The authors suggested CF livers may have a similar phenotype caused in KO1 mice. Do KO1 livers have altered CFTR expression or activity? If the study suggests the interaction of b-catenin and NF-κB with CFTR, KO1 could have altered CFTR expression and some phenotypes related to CFTR inhibition.This is an important point. What we are proposing is that loss of function mutations in CFTR leads to its inability to bind β-catenin in BECs which leads to its destabilization. The ensuing decrease in β-catenin protein allows for p65 to get activated more readily in the presence of cytokines to lead to NF-κB activation and downstream signaling. We do not believe that β-catenin loss impacts CFTR levels although we are unsure if that impacts CFTR function which needs to be investigated in the future. Since CFTR is expressed in BECs only, we used whole tissue lysate from WT1 and KO1 at baseline and at 6m recovery to test any changes in total CFTR levels. We did not find any changes in total levels of CFTR protein. This data has been included as supplemental figure 10 and text added in Results section and in the Discussion section as well.15. The authors should also provide histologic examination of liver injury in mice by HandE staining.We are including a histology image, 16. To study the repair of liver damage in KO1 and KO2 mice, it is necessary to determine whether KO1 and KO2 mice will spontaneously cause liver damage. Therefore, it is important to provide groups of KO1 and KO2 mice without CDE diet.WT2 and KO2 fed normal diet and without any CDE injury were assessed for serum biochemistry. Insignificant changes between these two genotypes in serum levels of ALT were observed. Intriguingly, there were a subset of KO2 mice maintained on normal diet and without any previous CDE diet induced injury, that showed higher levels of total bilirubin. These changes were insignificant as assessed by one way ANOVA. It is important to mention that KO2 fed CDE diet for 2 weeks and followed on normal diet for various times, begin to show repopulation of these livers by BEC-derived β-catenin-positive hepatocytes such that KO2 are no longer KOs. The serum biochemistry of these recovered mice is unremarkable and indistinguishable from WT2 and hence the comparison between KO2 on normal diet versus 2w CDE and normal diet recovery might not be necessary.Gastroenterology 131, 1561-1572 (2006)). In the current study, we examined the serum biochemistry from KO1 and WT1 mice on normal diet. We found insignificant differences in serum ALT and total bilirubin between WT1 and KO1 maintained on normal diet.KO1 and WT1 models have been published by us previously. We showed that there is no spontaneous injury but with increasing age (8 months), there is mild increase in serum BR . But addressing pathogenesis of steatosis is out of scope of the current study, which is mainly focused on examining the molecular basis of reactive ductules that leads to inflammation and fibrosis.18. Line 176, "KO2" should be "KO1".We apologize for this error and are grateful for pointing it out. This has been corrected.


Herein, we evaluated the importance of the FKN-CX3CR1 axis during IRI-related rejections using a mouse heterotopic heart transplantation model. FKN expression and graft survival was compared between wild-type C57BL/6 recipients transplanted with BALB/c hearts preserved for 8 (WT-IRI) and 0.5 h (WT-control) at 4°C. Graft survival of WT-IRI was shorter than that of WT-control. FKN was expressed on the vascular endothelium in WT-IRI allografts, but minimally in WT-control. The role of the FKN-CX3CR1 axis in IRI-related rejection was directly investigated using the transplant model with CX3CR1-deficient recipients (CX3CR1 KO-IRI) or treatment with anti-mouse FKN monoclonal antibodies. Graft survival of CX3CR1 KO-IRI was longer than that of WT-IRI; antibody treatment prolonged graft survival. The contribution of CX3CR1+ monocytes to IRI-related rejection was evaluated by adoptive transfer to CX3CR1 KO-IRI. Adoptive transfer of CX3CR1+ monocytes attenuated the effect of prolonged graft survival in CX3CR1 KO-IRI. Overall, the FKN-CX3CR1 axis plays a major role during IRI-related rejection; its blockade has the potential to improve the outcomes of deceased donor transplantation.Transplantation outcomes are affected by the increase in rejection associated with ischemia reperfusion injury (IRI). Fractalkine (FKN), a chemokine for recruitment of CX3CR1  Lengtejection , 15. TheFractalkine (FKN) is the only CX3C chemokine reported to date , 17. FKNIn contrast, FKN reportedly contributes to the pathological process of vascular and tissue injury in inflammation-mediated diseases and pathological conditions, including atherosclerosis, glomerulonephritis, rheumatoid arthritis, and transplant rejection, by enhancing migration and adhesion of CX3CR1-expressing leukocytes and promoting their transmigration to inflammatory sites , 27, 28.+ monocytes express CX3CR1  has not yet been fully clarified. We here assumed that FKN expression would be induced in grafts under long-term ischemia conditions and that monocytes infiltrated via the FKN-CX3CR1 axis have a significant impact on promotion of transplant rejection and graft failure. In the present study, we investigated the role of the FKN/CX3CR1 axis in a mouse model of IRI-related rejection using CX3CR1-deficient mice as recipients or an intervention with anti-mouse FKN neutralizing antibody (anti-FKN mAb), the emphasis being on the contribution of CX3CR1-positive monocytes.Male BALB/c and C57BL/6 mice were purchased from the Japan SLC Corporation . CX3CR1 homogenous knockout in C57BL/6 background mice was performed by KAN Research Institute . All mice were bred and maintained under specific pathogen-free conditions at the Institute of Laboratory at Tokyo Women’s Medical University . The pathogen-free conditions implemented were based on the criteria of the Central Institute for Experimental Animals . The Tokyo Women’s Medical University internal committee on the use and care of laboratory animals approved all experiments (Reference ID: AE19-081).®, Bristol-Myers Squibb, Lawrenceville, NJ, United States ) was administered intraperitoneally at a dose of 0.25 mg/day on the day of transplantation (day 0) and day 1. Graft engraftment was assessed by palpation with the presence of contraction as an indicator. Rejection was defined as complete cessation of contraction.All transplant procedures were performed under general anesthesia using sevoflurane. Fully vascularized ectopic heart grafts from BALB/c donors were transplanted into C57BL/6 or CX3CR1-deficient recipients using microsurgical techniques . To inveAfter perfusion fixation with 1% paraformaldehyde, heart grafts were collected and embedded with O.C.T. compound . The tissues were cut into 6 μm-thick sections, blocked with normal donkey serum, and stained with 10 μg/ml of goat anti-rat FKN antibody  and 50-fold diluted rabbit anti-mouse CD31 antibody . The combination of 500-fold diluted Alexa fluor 555-conjugated donkey anti-goat IgG (Abcam) and Alexa fluor 488-conjugated goat anti-rabbit IgG (Abcam) was employed as the set of secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).Grafts were procured at day 7 and hematoxylin-eosin staining was performed after fixation with 10% neutral phosphate-buffered formalin.Anti-FKN mAb (clone 5H8) and control IgG (anti-dinitrophenol mAb) , 31, 36 6/head on day−1.Bone marrows recovered from 10 week-old wild-type C57BL/6 mice were treated with ammonium chloride buffer for hemolysis, and cultured for 3 days at 37°C in RPMI 1640 medium supplemented with 50 ng/ml of recombinant mouse macrophage colony-stimulating factor (R and D Systems), 10% fetal bovine serum, 0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2–7.5), 1× MEM non-essential amino acid solution, 1 mM sodium pyruvate, 1× penicillin-streptomycin, and 100 μM 2-mercaptoethanol. Monocytes were isolated from post-culture cells using the CD115 MicroBead Kit . The isolated monocytes were transferred into CX3CR1-deficient recipients at 3×10™ II (BD Biosciences) and FlowJo software .To confirm the purity of CX3CR1-positive monocytes in the isolated cells, Fc blocking with anti-CD16/CD32 antibody  was followed by staining with phycoerythrin (PE)-conjugated mouse CX3CR1 antibody and fluorescein isothiocyanate (FITC)-conjugated CD115 . Anti-mouse CX3CR1 antibody (clone L2D11) was provided by KAN Research Institute. Flow cytometry analysis was carried out with a FACSCantop values <.05 were considered significant.Comparisons of graft survival were analyzed by the log-rank test using Prism seven software ; differences with p = .0303, log-rank test). Histopathological findings at day 3 and 7 showed more severe cell infiltration in the WT-IRI group than in the WT-control group  or 0.5 h (WT-control) and graft survival was compared between the two groups. As shown in ol group . These rp = .0226, log-rank test)  or 0.5 h (CX3CR1 KO-control) into CX3CR1-deficient recipients. As shown in nk test) . These rp = .0204, log-rank test). This result indicates that anti-FKN mAb treatment improves graft survival shortened by cold-ischemia conditions, confirming the importance of the FKN-CX3CR1 axis in IRI-related rejection. We compared the graft tissues at day 3 and 7 between the WT-IRI + anti-FKN mAb group  to those with control IgG treatment (WT-IRI + control IgG). As shown in Ab group  and the Ab group  and founAb group . These r6 of the isolated cells were transferred into CX3CR1-deficient mice. The next day, donor hearts preserved for 8 h at 4°C were transplanted into the mice (CX3CR1 KO-IRI + CX3CR1+ Mono). As shown in + Mono group (MST: 37.0 days) and the WT-IRI group (MST: 26.0 days). These results indicate that CX3CR1-positive monocytes are important effector cells in IRI-related rejection.We focused on CX3CR1-positive monocytes as effector cells essential for enhancing acute rejection of grafts subjected to prolonged cold ischemia. C57BL/6 bone marrow cells were cultured under M-CSF stimulation for 3 days, and monocytes were purified with anti-CD115 mAb. Flow cytometry analysis showed that more than 93% of the isolated cells were CX3CR1-positive monocytes . ApproxiPrevention and mitigation of the impact of IRI is critical for protection against graft dysfunction and improvement of graft survival if the grafts have been ischemic for a long-time following recovery from donors. For this purpose, regulation of immune cell infiltration in grafts during the initial process would be highly effective. The FKN-CX3CR1 axis plays an important role in immune defense by controlling the migration and adhesion of various types of immune cells involved in immune responses at inflammatory sites or infected areas. Furthermore, the FKN-CX3CR1 axis has been reported to be involved in the development of inflammation-associated diseases and pathological conditions. Using a murine ectopic transplantation model with hearts subjected to cold ischemia, the present study clarified the importance of the FKN-CX3CR1 axis and CX3CR1-positive monocytes in IRI-related rejection, and then demonstrated the potential of the FKN-CX3CR1 axis as a target for prevention of post-transplant graft dysfunction and rejection of long-term preserved grafts, such as those recovered from deceased donors.In the present study, we first analyzed the influences of cold ischemic time on grafts in a mouse heart transplantation model. The comparison between the grafts preserved for 8 h at 4 °C (IRI) and 0.5 h  showed that graft survival in the WT-IRI group was significantly shorter than that in the WT-control group , and mor−CD80/CD86 co-stimulatory signals in antigen presentation . As shown in + Mono and WT-IRI groups. Moreover, CX3CR1-deficient recipients without transferring monocytes (CX3CR1 KO-IRI) exhibited prolonged survival compared to the WT-IRI group  to confirm the infiltration of transplanted monocytes into graft tissues and early-stage histopathology. These rigorous analyses are necessary to further validate the role of the FKN-CX3CR1 axis in IRI-related rejection in the future.There are potential limitations to the present study. The following test have not been performed: 1) to compare the early-stage histopathology of the CX3CR1 KO-control, CX3CR1 KO-IRI and WT-IRI groups, 2) to compare the donor-reactive memory CD8In conclusion, ischemia in donor organs over time with respect to transplantation leads to exacerbation of graft rejection via impaired reperfusion after transplantation. CX3CR1-positive monocytes and the FKN-CX3CR1 axis play important roles in this series of tissue disorders that significantly affect allograft survival. Blockade of the FKN-CX3CR1 axis by anti-FKN antibodies or other means reduces the impact of IRI-related rejection and could be an effective intervention to improve the outcomes of deceased donor transplantation.This study evaluated the significance of the FKN-CX3CR1 pathway during ischemia-reperfusion injury (IRI)-related graft rejections using a mouse heterotopic heart transplantation model. We believe that our study makes a significant contribution to understanding the mechanism because the roles of the FKN-CX3CR1 axis in IRI-related rejection were directly investigated using the transplant model with CX3CR1-deficient recipients (CX3CR1-KO IRI) or treatment with anti-mouse FKN monoclonal antibodies. Our findings indicated that the FKN-CX3CR1 axis plays a major part during IRI-related rejection and its blockade has the potential to improve the outcomes of deceased donor transplantation. Further, we believe that this paper will be of interest to the readership of your journal because it is known the outcome of transplantation is affected by the promotion of rejection associated with IRI. Our findings suggest there is a way to potentially mitigate this phenomenon and enhance the acceptance of graft transplantations from deceased donors.


Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires’ disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila. Legionella pneumophila is the causative agent of a life-threatening pneumonia called Legionnaires’ disease. By infecting a broad range of protozoa in the environment, L. pneumophila has acquired distinct but overlapping mechanisms of virulence that rely on over 300 secreted effector proteins -buffered yeast extract (AYE) broth or on buffered charcoal yeast extract (BCYE) agar [L. pneumophila grown on solid media were incubated aerobically at 37 °C for 72 h, while broth cultures were grown overnight with shaking. Escherichia coli cultures were grown in Luria-Bertani (LB) broth or agar. When necessary, ampicillin, kanamycin, and/or chloramphenicol were added to LB broth or agar at 100 μg/mL, 100 μg/mL, and 12.5 μg/mL, respectively. E. coli grown on solid media or in broth were incubated aerobically at 37 °C overnight. Yeast strains were grown at 30 °C in yeast extract/peptone/dextrose (YPD) medium or yeast nitrogen minimal medium supplemented with 2% glucose and amino acids including histidine (20 mg mL−1), methionine (20 mg mL−1), tryptophan (20 mg mL−1), adenine (20 mg mL−1), uracil (20 mg mL−1), and leucine (30 mg mL−1), when necessary. The bacterial strains, yeast strains, and plasmids used in this study are listed in YE) agar . Antibio®, Waltham, MA, USA). Heat inactivated HyCloneTM Foetal Bovine Serum  and penicillin/streptomycin were added at 10% and 1% of the concentration, respectively. All cell lines were grown in T75 cm2 flasks , incubated in 5% CO2 at 37 °C, and split at a cell-media ratio of 1:10 after 48–72 h growth.Human embryonic kidney 293T cells (HEK293T) expressing Fcγ-receptor (HEK-293 Fcrγ) and HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX-1  and supplemented with 10% heat-inactivated Fetal Bovine Serum (Gibco). The human monocytic cell line, THP-1, was grown in RPMI 1640 medium GlutaMAX  (primers MavL(pTEM1)F and MavL(pTEM1)R), before cloning corresponding product into pXDC61 harboring-TEM-1. Cell transfection constructs expressing GFP-tagged MavL and FLAG-tagged UBE2Q1 were generated through amplification of mavL (primers MavL(pGFP)F and MavL(pGFP)R) and ube2q1 (primers UBE2Q1(pFLAG)F and UBE2Q1(pFLAG)R), before cloning corresponding products into pEGFP-C2 and p3xFLAG-Myc-CMV™. To create plasmid p4HA-MavL expressing HA-tagged MavL during Legionella infections, mavL was amplified using primers MavL(p4HA)F and MavL(p4HA)R, and the corresponding product was cloned into pMMB207c. To create plasmid pGBKT7-MavL for yeast two-hybrid screening, mavL was amplified using primers MavL(pGBK)F and MavL(pGBK)R, and the corresponding product was cloned into pGBKT7. The plasmids and primers used in this study are listed in E. coli through transformation of plasmid DNA into chemically competent XL1-Blue via heat-shock, in accordance with the protocols of the manufacturer. Plasmids were extracted using the AxyPrep™ plasmid DNA purification miniprep kit . Transformation of plasmid DNA into L. pneumophila was performed via electroporation. Overnight cultures of L. pneumophila were grown in AYE broth, pelleted in PBS, washed with ice-cold water, and resuspended in water +10% (v/v) glycerol. Plasmid DNA was then added and L. pneumophila cells pulsed at 2.3 kV, before cells were grown in AYE broth and subsequently plated on BCYE with appropriate antibiotics for 48 h. Transformation of plasmid DNA into the yeast strain AH109 (MATa) was performed according to the lithium acetate method [Plasmid constructs were maintained in e method , and tra4 cells/well in 96-well tissue culture plates (Corning), phorbol-12 myristate 13-acetate (PMA) was added (10−8 M) to stimulate the differentiation of THP-1 monocytes into macrophages, and the cells were incubated for 24 h. L. pneumophila strains were grown in AYE broth with 1 mM IPTG and 12.5 µg/mL chloramphenicol. Cells were inoculated in triplicate with L. pneumophila strains (MOI of 1:40) for 1 h. Cells were then washed with Hanks’ Balanced Salt Solution (HBSS) supplemented with 5% (v/v) HEPES. Beta-lactamase-loading solutions  and CCF2-AM substrate  were added with 2.5 mM probenecid, in accordance with the protocols of the manufacturer. Cells were incubated at room temperature for 105 min before loading solutions were removed and replaced with HBSS + 5% HEPES + 2.5 mM probenicid. A ClarioSTAR microplate reader  was then used to detect emissions at 450 nm and 520 nm after samples were excited with transmission of 410 nm light. The ratio of 450–520 nm light was calculated, and background fluorescence was subtracted and normalised against negative controls. An unpaired two-tailed Student’s t-test was applied to determine significance. At least three biological repeats of the translation assay were performed. THP-1 cells were seeded at 8 × 10L. pneumophila protein MavL as bait with the Matchmaker pre-transformed normalised HeLa cDNA library , in accordance with the instructions of the manufacturer (Clontech PT3183-1). In this Y2H assay, the yeast strain AH109 was transformed with pGBKT7-mavL using the LiAc method and mated with the yeast strain Y187M pre-transformed with the pGADT7 RecAB plasmid carrying the HeLa cDNA library. Positive clones were selected by plating on quadruple dropout (QDO) media . Clones were also plated on low stringency or double dropout (DDO) SD media (SD/-Leu/-Trp), as a selection of only diploids with both prey and bait plasmids. Colonies were re-streaked on QDO, to ensure expression of reporter genes and, therefore, positive interaction. Colonies that were able to grow on both DDO and QDO plates were considered true positives, whereas colonies that could only grow on DDO plates were deemed false positives. The pGADT7-Rec-cDNA plasmids were isolated using the ZymoprepTM Yeast Plasmid Miniprep I Kit , transformed into XL-1 Blue cells, and plated onto LB agar supplemented with ampicillin. The pGADT7-Rec-cDNA plasmids were then extracted and sequenced to identify the cDNA inserts.The yeast two-hybrid (Y2H) screen was performed using lacZ reporter. Briefly, pairwise bait and prey plasmids  were transformed into S. cerevisiae strain AH109 using the LiAc method [− and Leu− plates and were grown overnight (OD600 of 0.6) before lysis in Z-buffer  and being assayed for the level of β-galactosidase activity using Ortho-nitrophenyl-p-D-galactopyraniside (ONPG) as a substrate. Data are from at least three biological repeats performed in triplicate. Quantitative β-galactosidase assays were performed, in accordance with the protocols of the manufacturer , to measure the expression of the Y2HS c method . The yea® 6 , and incubated for 20 h. Cells were then washed 3 times with PBS before adding 600 µL KalB lysis buffer  supplemented with 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF and Complete Protease Inhibitor . Transfected cells were then pelleted and supernatants collected. Anti-FLAG M2 monoclonal magnetic beads  were added to the cell lysate supernatant, in accordance with the protocl of the manufacturer, and incubated on a rotating wheel at 4 °C for approximately 24 h. Cell lysate was removed and FLAG beads were washed 3 times with lysis buffer. FLAG peptide  was added to the beads at a concentration of 100 μg/mL and incubated for 30 min at 4 °C to elute FLAG-tagged proteins. Immunoprecipitation experiments were performed during at least three biological repeats.HeLa cells were seeded in 10 cm tissue culture dishes. After 24 h, cells were transfected with 11 µg of purified p3xFLAG-Myc-CMV™-24 or pFLAG-UBE2Q1, plus pEGFP-Lem26 or pEGFP-MavL using FuGENEw/v) milk power and 0.1% Tween-20  for 1 h. After washing with TBS + 0.1% Tween-20, primary antibodies were applied overnight in TBS + 0.1% Tween-20 + 5% BSA at 4 °C. Primary antibodies used were: anti-FLAG-HRP conjugate  at 1:2000, anti-FLAG (Sigma-Aldrich) at 1:2000, anti-β-actin at 1:4000 (AC-15), anti-beta lactamase  at 1:1500, and anti-GFP at 1:2000 . After further washing, secondary antibodies were then applied for 1 h. HRP-conjugated rabbit α-mouse and goat α-rabbit  antibodies were applied and were appropriate at 1:2000 dilutions in TBS + 0.1% Tween-20 + 5% BSA. Development reagents  were applied to membranes, in accordance with the protocols of the manufacturer, and chemiluminescence was then visualised using a ChemiBIS imaging system . Samples were prepared for SDS-PAGE gel electrophoresis, with the addition of lithium dodecyl sulphate (LDS) sample buffer and dithiothreitol (DTT) reducing agent, and heated to 70 °C for 10 min. Samples were then loaded onto NuPAGE™ 4–12% bis-tris gels (Life Technologies) and separated via electrophoresis at 165 V for 30 min in MES buffer (Life Technologies). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the iBLOT-2™ transfer system (Life Technologies). After transfer, membranes were placed in a blocking solution of tris-buffered saline (TBS) with 5%  to induce protein expression. Prior to infection, the L. pneumophila was diluted to a concentration of 108 CFU/mL and coated with rabbit polyclonal anti-L. pneumophila  at 37 °C in 5% CO2 for 20 min prior to opsonization at an MOI of 1. Cells were infected at a multiplicity of infection of 10 for 16 h. Cells were then fixed in 4% paraformaldehyde-PBS for 20 min, then treated with 0.1% TritonX-100-PBS for 20 min and blocked with 3% BSA-PBS for 30 min. Cells were then incubated for 60 min in staining solution containing 0.2% BSA and a 1:50 dilution of anti-HA.11 monoclonal antibody  for the detection of HA-fusions and a 1:50 dilution of anti-Legionella polyclonal antibody (Meridian Life Science), before the bound primary antibody was detected using a 1:1000 dilution of Alexa Fluor 488-conjugated anti-mouse antibody and Alexa Fluor 568-conjugated anti-rabbit antibody. For the last 10 min, HOECHST stain  was added to the staining solution. Coverslips were mounted onto glass slides with Dako Fluorescent Mounting Medium . Immunofluorescence images were acquired using a Zeiss confocal laser-scanning microscope with a 100×/EC Epiplan-Apochromat oil-immersion objective. To determine effector subcellular localization during lpg2526) was cloned into pMCSG7 and pRL652 vectors, by ligation independent cloning (LIC). Initially, a construct consisting of residues 2-444 was designed. This construct expressed and purified poorly, compelling us to explore alternative constructs. The PsiBLAST algorithm indicated a start site mis-annotation for MavL; most homologues had start sites corresponding to the MavL residue M41. Two constructs of MavL were designed to investigate this putative start site, namely MavL(42-435) and MavL(42-388). Inserts corresponding to these sequences were amplified from the MavL(2-444) plasmid and inserted into pMSCG7 and pRL652 by LIC.The gene encoding MavL  glycerol, 0.1% (v/v) Triton X-100) and lysed two times at 35 kPsi in a cell disruptor . The lysate was spun at 21,000× g for 30 min in a Beckman JA25.50 rotor. Supernatant was added to 5 mL of Qiagen Ni-NTA beads pre-equilibrated with three column volumes of a buffer containing 20 mM Tris, pH 8.0, 50 mM NaCl and the beads were washed with 50 mL of this buffer. Protein was eluted with the same buffer supplemented with 100 mM imidazole. Purified protein was concentrated to 16 mg/mL in a 10 kDa molecular weight cut-off Millipore centrifugal filter span at 4000× g. Finally, the protein was loaded onto a Biorad SEC650 or S200 Increase  size exclusion column for separation of monomeric from dimeric species.MavL(42-435) and MavL(42-388) in pMCSG7 were first transformed into BL21(DE3)pLysS and plated on LB agar containing ampicillin (100 µg/mL). A single transformant was inoculated into 20 mL of LB supplemented with ampicillin (100 µg/mL) and glucose (0.4%), and grown overnight at 37 °C. This culture was then sub-cultured into 1 L of terrific broth (TB) supplemented with ampicillin (100 µg/mL) and grown at 37 °C until an optical density (A600) of ~1.0 had been achieved, at which point the temperature was reduced to 18 °C and 500 µM of Isopropyl β-D thiogalactopyranoside (IPTG) was added to the culture to induce protein expression. The cells were then incubated for approximately 16 more hours before being pelleted at 6900× Limited proteolysis on MavL(42-435) and MavL(42-388) showed the latter construct to have greater susceptibility to degradation than the former. Furthermore, crystals were obtained only for a dimer species of MavL(42-435). This MavL(42-435) construct was, therefore, used in all downstream structural and ligand-binding studies.Purification of MavL(42-435) on a Biorad SEC650 or GE S200 column allowed for the separation of dimer from monomer species, which were then concentrated to 15–20 mg/mL for crystallization trials. Initially, MavL(42-435) eluted from size exclusion predominantly in the monomeric form. The dimer species became more prominent after a day at 4 °C. By the third day, the dimer peak was larger than that of the monomer. At this point, further incubation of MavL(42-435) at 4 °C no longer increased the presence of the dimer. Re-running monomeric MavL on gel filtration regenerated some dimer and vice versa. These findings show that monomer and dimer species of MavL(42-435) are in equilibrium, with the monomer favored initially.Intriguingly, only the MavL(42-435) dimer was found to crystallize. This observation comes in conflict with the previously reported equilibrium between monomer and dimer species. To account for this, it is possible that mixing purified MavL(42-435) with the crystallization solution stabilized the dimer. If so, rapid screening of the dimer after separation would have been essential to the crystallization of MavL(42-435). Consistent with this notion, screening of MavL was always carried out immediately after purification and any protein that could not be screened was flash-frozen in liquid nitrogen. In the few cases where protein was left at 4 °C, it gradually lost its crystallisability. E. coli competent cells. These transformants grew well in minimal media supplemented with methionine, but not without it. Induction of protein expression was carried out as described previously, using 500 µL IPTG and reducing the temperature to 18 °C. Importantly, the culture was resuspended in minimal media lacking methionine immediately prior to induction, at which point seleno-methionine was added to the culture. In this way, the incorporation of seleno-methionine into recombinant protein was ensured. Purification of seleno-methionine (SeMet) MavL(42-435) was carried out, as described for native MavL(42-435).No phasing model for MavL(42-435) was present in the protein data bank (PDB). We, therefore, opted to produce a seleno-methionine derivative for phasing by single anomalous dispersion (SAD). Inhibition of methionine biosynthesis was initially attempted in BL21(DE3)pLysS cells harboring the MavL(42-435) plasmid in pMCSG7. We found that growth of these cells in minimal media was prohibitively impaired. To overcome this, the MavL(42-435) plasmid was transformed into auxotrophic B834 v/v Polyethylene glycol 400, 2.0 M Ammonium sulfate) produced poorly diffracting needle clusters in less than three days of growth. Extensive efforts to optimize the morphology of these crystals using additive- and grid-screens were met with little success. A few months later, an improved crystal form of MavL(42-435) was discovered in Crystal Screen condition B3 . These slower growing crystals diffracted better than the initial needle-clusters. After optimization, the best crystals were obtained at 20 °C in drops containing 1 µL protein in 15 mM Tris-HCl, pH 8.0, 50 mM NaCl mixed with 1 µL reservoir comprised of 0.2 M Ammonium sulfate, 0.1 M Citrate pH 6.0, and 22% w/v Polyethylene glycol 8000. Both native and seleno-methionine derivative MavL produced optimal crystals in this condition.Initial crystals for MavL(42-435) were obtained from Crystal Screen HT . Specifically, conditions A4  and D3  glycerol. Diffraction data was collected to 2.65 Å at the Canadian Macromolecular Crystallography Facility (CMCF) 08ID beamline, Canadian Light Source (CLS), using a Pilatus3 S 6M detector [Seleno-methionine derivative MavL crystals were transferred into 1 µL mother liquor containing 25%  and analyzed with the NANOANALYZE software . Both ligand and protein were in a buffer containing 15 mM Tris-HCl, pH 8.0, and 50 mM NaCl. The experiment was carried out at 20 °C.L. pneumophila effector proteins is their translocation into the host cell via the Dot/Icm secretion system. Many Dot/Icm effectors then mimic host cellular machinery using eukaryotic-like domains [L. pneumophila infection [L. pneumophila 130b; an isogenic ΔdotA mutant. L. pneumophila 130b carrying pTEM1-RalF was included as a positive control while ΔdotA pTEM1-RalF was included as negative a control  for 16 h. Immunostaining with anti-HA antibodies showed that MavL staining occurred throughout the host cytoplasm but was excluded from the nucleus  harbouring pGBKT7-mavL alone could not grow on quadruple knockout (QDO) selective yeast minimal media (YMM) . Screening for MavL eukaryotic interacting partners recovered approximately 500 positive prey clones. Sequencing of rescued cDNA plasmids from 50 randomly selected yeast colonies yielded only one MavL binding partner, the E2 conjugating enzyme, UBE2Q1 . The interaction between MavL and UBE2Q1 was confirmed by re-transforming pGBKT7-mavL and pGADT7-UBE2Q1 into S. cerevisiae strain AH109 and plating on selective media. S. cerevisiae growth was only observed on QDO YMM plates when both plasmids were present, supporting the interaction between MavL and UBE2Q1  of MavL, we performed a yeast two-hybrid (Y2H) screen using a HeLa cDNA library as prey and full-length MavL as bait. Prior to screening the library, we confirmed that MavL did not auto-activate reporter gene expression. This was done by ensuring that d UBE2Q1 A. Additid UBE2Q1 B. Yeast Next, we aimed to observe MavL interacting with UBE2Q1 in mammalian cells via co-immunoprecipitation (co-IP). HeLa cells were transfected with 3xFLAG-tagged UBE2Q1 and GFP-tagged MavL for 20 h, and co-IP was performed using whole cell lysates. The cell lysates were precipitated with anti-FLAG magnetic beads and analyzed by immunoblot using anti-FLAG and anti-GFP antibodies. GFP-MavL (70.7 kDa) was detected in the immunoprecipitate of cells co-transfected with 3xFLAG-UBE2Q1 (47.75 kDa), but not when co-transfected with 3xFLAG empty vector (0.8 kDa). In addition, an unrelated effector protein, GFP-Lem26 (112.19 kDa), was not detected after co-transfection and co-IP with 3xFLAG-UBE2Q1 C. This rTo learn more about the biochemical function of MavL, the crystal structure of His-MavL(42-435) was solved by single anomalous dispersion (SAD) and refined to a resolution of 2.65 Å. This construct starts from the alternative start at Met41 and does not contain the 20 C-terminal residues, which are predicted disordered and contain the putative E-bloc translocation signal. There are three molecules in the asymmetric unit. They are nearly identical and superimpose with a root-mean-square deviation (rmsd) of ~0.4 Å. The entire molecule forms a single domain. The core of each molecule is formed by a highly curved, twisted, ten-stranded mixed β-sheet A. Two α-The level of amino acid conservation along the MavL sequence was determined by sequence alignment with protein sequences from UniProt  and projA quest for proteins with a similar fold to MavL was conducted using the DALI server ,23. LimiA closer examination of the superpositions with the above-mentioned proteins suggests that the most likely functional homologs of MavL are the macrodomains. Therefore, next we performed a detailed comparison of MavL with various proteins containing macrodomains. The common core of ADPR-binding macrodomains contains ~200 residues and is an α/β/α sandwich with a curved central six-stranded mixed β-sheet covered on the convex side by three long α-helices and with two α-helices lining the concave face of the β-sheet. Most macrodomains contain additional α-helices and/or β-strands ,30,31,32Thermomonospora curvata (PDB ID 3SIG [T. curvata macrodomain the order of strands is ↑β2-↑β7-↑β6-(α2)-↑β3-↑β5-(α1)-↓β4-↑β1. The order of the structurally corresponding strands in MavL is different, namely ↑β6-↑β5-↑β4-(α2)-↑β7-↑β3-(α1)- ↓β2-↑β1  glycohydrolase from  ID 3SIG , which c ↓β2-↑β1 C. The N- ↓β2-↑β1 A. Thus,  ID 4EPP , the ADPWith these overall structural similarities between MavL and macrodomains in mind, as well as the presence of a deep cleft capable of accommodating ADPR, we next tested experimentally if MavL can bind ADP-ribose and, if so, what the binding constant would be. We have applied isothermal titration calorimetry (ITC) to test this hypothesis. The binding was easily detected by ITC and the binding constant Kd was estimated to be 13 µM , support+ and modify target proteins on a specific amino acid. There are two classes of ARTs with similar fold, cholera toxin-like ARTCs, containing R-S-F-E motif and diphtheria toxin-like ARTDs containing H-Y-Y/F-E motif [ADP-ribose binding proteins can be classified as writers, readers, or erasers ,35. Writ-E motif ,37. Read-E motif . We carrMavL does not superimpose with the C-terminal catalytic writer domain of PARP14/ARTD8. This is not altogether surprising, as ARTD writers adopt a different overall fold than macrodomain readers. Briefly, writers contain an antiparallel twisted β-sheet sandwich flanked by several α-helices t ,40. MavL6-8-QEE motif within their ligand-binding site that facilitates catalysis [Thermomonospora curvata PARG (PDB ID 3SIG) has been proposed, where the side-chain carboxyl of a glutamate from the conserved motif (Glu115) is deprotonated by the glycosidic oxygen that links ribose moieties in the chain [T. curvata PARG [T. curvata PARG, the reader macrodomains 2 and 3 of multidomain PARP14  lack acidic residues corresponding to Glu114-Glu115 of PARG. Furthermore, Phe227 in PARG is replaced by a Leu1137 in PARP14, and no cluster of glycine residues is present in the corresponding position of PARP14, which results in a somewhat different orientation of the ribose moiety in these two binding sites. Thus, although ADP-ribose reader macrodomains and glycohydrolases share structurally similar ligand binding pockets, they can be distinguished by the presence/absence of the GGG-X6-8-QEE motif. Since MavL shares fold similarity with ADP-ribose-binding macrodomains, it is important to consider readers and erasers. Erasers differ from readers by a unique GGG-Xatalysis ,38,41. Ahe chain . This lehe chain . Three gata PARG ,41. In c6-8-QEE motif characteristic of eraser macrodomains, however, it contains an arrangement of acidic residues that are spatially equivalent to the acidic cluster in eraser macrodomains. Specifically, QE114E115 of T. curvata PARG (3SIG) superimposes with D332D333G in MavL, and the carboxylic groups of Asp333 and Glu114 are in close proximity. Moreover, the GGG of PARG are replaced by Gly321-Asn322-Asp323, with the acidic group of Asp323 assuming similar position to the catalytic Glu115 in PARG 3SIG  (data not shown). However, in contrast to the human PARG control, MavL was unable to hydrolyze the ADP-ribose moieties attached to PARP1. This result indicates that MavL does not act as a PARG for poly-(ADP-ribosylated) PARP1. However, MavL could recognize a different ADP-ribosylated protein substrate, and further studies will be required to validate the glycohydrolase activity of MavL and to identify the host cellular target of this activity.MavL does not contain the GGG-XARG 3SIG . Additio6-8-QEE) lining the ADP-ribose binding pocket. MavL lacks this specific motif, however, it contains several aspartates in the same part of the ADP-ribose binding site, with their acidic groups in similar positions to the two glutamates from the macrodomain eraser motif. The different connectivity of β-strands and placement of putative active site residues in MavL, while leading to the formation of a similar ADP-ribose binding site to macrodomains, strongly suggests a unique evolutionary history for MavL relative to the classical macrodomains, converging toward the same function. The crystal structure of MavL(42-435) reveals partial fold similarity to several classes of enzymes that contain a mixed β-sheet flanked on one side by two long α-helices. The most likely similarity of functional relevance is to ADP-ribose-binding macrodomains. Interestingly, while the spatial localization of several central β-strands and two α-helices overlap, the connectivity of the secondary structure elements along the primary sequence in MavL differs from the classic macrodomains. Macrodomains function either as readers or erasers. The latter have a characteristic ADP-ribosyl glycohydrolase motif  glycohydrolase (PARG) activity. Initial experiments to establish an ADP-ribosyl glycohydrolases activity using poly-(ADP-ribosylated) PARP1 as a substrate showed no detectable activity. Despite the similarity between MavL and eraser enzymes, these findings may point to a role for MavL as an ADP-ribose reader rather than eraser. If MavL does indeed have eraser activity, it could compensate for the effect of a bacterial or eukaryotic writer present in the host cell. Aberrant ADP-ribosylation can have severe consequences for the host cell, including the inhibition of protein synthesis or overproduction of cAMP. Many species of pathogenic bacteria have exploited this powerful modification using secreted effectors that exhibit mono- or poly-ADP-ribosyltransferase (MAR/PAR) activity. Indeed, the SidE family of Dot/Icm effectors rely extensively on ADP-ribosylation to ubiquitinate host proteins in an all-in-one approach. SidE effectors contain mono ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains that act in concert to facilitate host protein ubiquitination. Initially, an ADP-ribose moiety is transferred to ubiquitin by the mART domain of SidE. Activity of the PDE domain then renders phosphoribosyl-ubiquitin, which is finally attached to a serine residue on the target protein. L. pneumophila effector Lem26 (Lpg2523), encoded by a gene located in proximity to mavL, is a possible candidate for a writer as Lem26 contains a predicted phosphodiesterase (PDE) domain, which catalyzes the conjugation of ADP-ribosylated ubiquitin (ADPR-Ub) to a serine residue on substrates; Lem26 also causes a growth defect in yeast that is significantly reduced in the presence of MavL, suggesting a meta-effector relationship [Interestingly, the tionship . Moreovetionship , they maWe found that MavL binds the E2 ubiquitin-conjugating enzyme, UBE2Q1, by yeast two-hybrid and co-immunoprecipitation. To rationalize why MavL binds UBE2Q1, it is convenient to imagine that Lem26 acts a writer and MavL acts as an eraser enzyme. If Lem26 or another host protein catalyzes the addition of ADP-ribose onto ubiquitin that is in complex with UBE2Q1, MavL could reverse this modification using eraser activity. In such a case, we should only expect MavL to bind ADP-ribosylated UBE2Q1 in a cellular context in vivo. It should be noted, however, that the function of Lem26 remains unknown and there is presently little published data on this protein. Still, the data presented here may point to meta-effector activity between MavL and Lem26, which warrants further exploration of the roles these proteins play in ADP-ribosylation and ubiquitination.In summary, this study provides crystallographic and binding data that point to a putative role for MavL in ADP-ribosylation. Structural comparisons of MavL with ADP-ribose-binding macrodomains suggest reader or eraser activity against mono-ADP-ribosylated substrates. Although the biological role of MavL remains unclear, the studies presented here will hopefully guide further research toward this end.


Epithelial-mesenchymal transition (EMT) is defined as a process in which differentiated epithelial cells undergo phenotypic transformation into myofibroblasts capable of producing extracellular matrix, and is generally regarded as an integral part of fibrogenesis after tissue injury. Although there is evidence that the complete EMT of tubular epithelial cells (TECs) is not a major contributor to interstitial myofibroblasts in kidney fibrosis, the partial EMT, a status that damaged TECs remain inside tubules, and co-express both epithelial and mesenchymal markers, has been demonstrated to be a crucial stage for intensifying fibrogenesis in the interstitium. The process of tubular EMT is governed by multiple intracellular pathways, among which Wnt/β-catenin signaling is considered to be essential mainly because it controls the transcriptome associated with EMT, making it a potential therapeutic target against kidney fibrosis. A growing body of data suggest that reducing the hyperactivity of Wnt/β-catenin by natural compounds, specific inhibitors, or manipulation of genes expression attenuates tubular EMT, and interstitial fibrogenesis in the TECs cultured under profibrotic environments and in animal models of kidney fibrosis. These emerging therapeutic strategies in basic researches may provide beneficial ideas for clinical prevention and treatment of chronic kidney disease. Kidney fibrosis, a pathological process characterized by excessive deposition of extracellular matrix (ECM) in the interstitium accompanied by destruction of normal kidney architecture, is a hallmark and inevitable end point of all kinds of progressive chronic kidney disease (CKD). Myofibroblast is well known as the major type of matrix-producing cell, the source of which has long been controversial and remains a hot area of research in nephrology . Based oTEC, as a major component of renal parenchyma, is particularly vulnerable to damage during acute kidney injury (AKI) and is also a driving force for the progression of CKD. The involvement of epithelial-mesenchymal transition (EMT) in kidney fibrosis is widely concerned . EMT is EMT process is governed by sophisticated signal networks involving several developmental pathways, such as Wnt, Notch, and Hedgehog. Of them, the role of Wnt/β-catenin signaling is believed to be essential. Numerous studies have demonstrated that Wnt/β-catenin signaling is a potent mediator of EMT process. Hence, the intervention in EMT via modulating activity of this pathway is considered a promising therapeutic strategy against kidney fibrosis. In this mini review, we briefly discuss the mechanisms by which Wnt/β-catenin signaling regulates tubular EMT process, and summarize current strategies to interfere with EMT by modulating activity of this signaling.In canonical Wnt cascade, when Wnt ligands bind to receptors Frizzled protein (FZD) and lipoprotein receptor-related protein-5 or 6 (LRP5/6), Disheveled protein (Dvl) is recruited and a cytoplasmic destruction complex comprising proteins adenomatous polyposis coli (APC), Axin, casein kinase 1 (CK1), and glycogen synthase kinase 3β (GSK3β) is inhibited, resulting in de-phosphorylation, stabilization, and nuclear translocation of β-catenin. In the nucleus, the combination of β-catenin with T-cell factor and lymphoid enhancer-binding factor (TCF/LEF) initiates transcription of Wnt target genes . Wnt/β-cTECs are a main source of Wnt proteins in injured kidneys, and these ligands act in an autocrine or paracrine manner between several cell types. Activation of Wnt/β-catenin signaling induces transformation of TECs into a secretory phenotype with most partial EMT and a few complete EMT, induces proliferation, activation and differentiation of interstitial fibroblasts into myofibroblasts, and induces polarization M2 phenotype, and pro-inflammatory activation of macrophages. In turn, Wnt ligands derived from fibroblasts and macrophages can also target TECs directly .In TECs, Wnt/β-catenin signaling takes effects by inducing its target genes, some of which play the substantial roles in regulating tubular EMT process in the setting of CKD, such as fibroblast-specific protein 1 (FSP-1), fibronectin, matrix metalloproteinase 7 (MMP7), Snail, and Twist . ConcretSnail is a member of the zinc finger 1 transcription factor family and is able to trigger the first step in EMT process by transcriptionally suppressing the expression of E-cadherin and disrupting adhesions between epithelial cells . By reprTwist is a transcription factor of the basic helix-loop-helix class and is capable of not only repressing E-cadherin gene transcription by binding to the E-boxes in its promoter region but also inducing the expression of mesenchymal markers including fibronectin, vimentin, α-SMA, and N-cadherin . AdditioIn a short, sustained activation of Wnt/β-catenin signaling is a potent propeller of EMT. Therefore, it represents a promising therapeutic target to restrain tubular EMT process and mitigate kidney fibrosis.A great deal of strategies for hampering tubular EMT process and alleviating kidney fibrosis through inhibiting the activity of Wnt/β-catenin signaling in various animal or cellular models of CKD have been reported, in which the components of Wnt/β-catenin pathway were selectively or specifically detected for exploring the intrinsic relationship between the strategy and the change in the activity of the signaling. These studies are shown below based on the most upstream level of the components of the signaling pathway being examined and are summarized in in vivo and in vitro (β-Arrestin-1 is a negative adapter of G-protein-coupled receptors (GPCRs) and also acts as a scaffold protein that regulates various cellular functions independently of GPCR activation . Xu et ain vitro .Advanced oxidative protein product (AOPP), belonging to dityrosine-containing protein family, is a marker of protein glycoxidation closely related to oxidative stress. As a uremic toxin, AOPP has been found accumulation in patients with CKD. Chronic accumulation of AOPP aggravated kidney fibrosis in animal models . Feng etSalt inducible kinase 1 (SIK1), a member of AMP-activated protein kinases (AMPKs) family, plays a key role in regulating metabolism, cell survival, and growth . Hu et aExtracellular signal-regulated kinase-1 and 2 (ERK1/2) are serine/threonine kinases that have been found to be involved in uric acid-mediated EMT and the pathogenesis of hyperuricemic nephropathy (HN) . Tao et MMPs are a family of zinc-dependent proteases, and MMPs-mediated destruction of tubule basement membrane integrity was once believed to be a key step in promoting EMT . AlthougAlismatis rhizoma, a natural product with lipid-lowering and renoprotective effects , a saponin extracted from otection . Wang et complex .Rhizoma Atractylodis macrocephalae, possesses various biological activities including antioxidant and anticancer (Atractylenolide I (ATL-1), a eudesmane-type sesquiterpenoid lactone derivative of ticancer . Guo et ticancer .Wnt-inducible signaling pathway protein-1 , belonging to the CCN family of ECM proteins, is a downstream target of Wnt/β-catenin, and has been shown to be involved in fibrotic diseases . Chen etin vivo (C-reactive protein (CRP), an acute phase plasma protein, is generally considered as a non-specific marker of inflammation , howeverin vivo .in vivo , also known as chemokine (C-X3-C motif) ligand 1, is a chemokine that regulates cell adhesion and growth and has been shown to be involved in the pathogenesis of inflammatory diseases including autoimmune disease . Fu et ayclin D1 .R-spondin1 (RSPO1), a member of secretory protein RSPOs family, possesses a high affinity with leucine-rich repeat-containing G protein coupled receptor 4 (LGR4). RSPO1 has been identified as an activator of Wnt/β-catenin signaling because the binding of LGR4 and RSPO1 enhanced Wnt-induced phosphorylation of LRP6 . It has − channel, is abundantly expressed at the apical surfaces of proximal, and distal tubules in normal kidneys , a cAMP-activated Cl kidneys . Zhang eactivity .Dedicator of cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor for Rac, has been reported to enhance the stability and activity of β-catenin and induce EMT by interacting with β-catenin degradation complex to increase the level of cellular β-catenin response to Wnt ligands . Zhu et AMPK, a heterotrimeric serine/threonine protein kinase, functions as an energy sensor in response to stresses, and regulates cell energy balance and differentiation . Qiu et Four and a half LIM domain protein 2 (FHL2) belongs to the members of FHL subfamily that is included in LIM-only proteins family. FHL2 acts as a scaffold protein interacting with various intracellular protein partners, enabling it to regulate signaling pathways that involve a plethora of cellular tasks . We haveAlthough TECs undergoing conventional EMT are no longer recognized as a major constituent of interstitial myofibroblasts, the partial EMT has been demonstrated to exert the crucial functions in the fibrogenesis during kidney fibrosis progression. Given the importance of Wnt/β-catenin signaling in the regulation of EMT, targeting this pathway to restrain tubular EMT process has become a promising strategy for inhibiting kidney fibrosis, which has attracted numerous researchers to conduct relevant studies.Data from animal models and cell experiments suggest that inhibiting Wnt/β-catenin signaling activity by either natural compounds, specific inhibitors, or manipulation of selective genes expression, may effectively suppress tubular EMT process and mitigate kidney interstitial fibrosis. Some of these studies have deeply investigated the mechanism by which Wnt/β-catenin activity is inhibited, while others have only observed the inhibitory effect of certain modulators on Wnt/β-catenin activity but not the mechanism of the signaling activity inhibition. Potential targeted components of Wnt/β-catenin pathway by various modulators are summarized in