Down-regulation of STAT3 enhanced chemokine expression and neutrophil recruitment in biliary atresia.
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ABSTRACT:
SUBMITTER: Fu M
PROVIDER: S-EPMC8035628 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundTissue infiltration by neutrophils during acute inflammatory states causes substantial tissue injury. While the magnitude of tissue neutrophil accumulation in innate immune responses is profoundly greater in males than females, fundamental aspects of the molecular mechanisms underlying these sex differences remain largely unknown.MethodsWe investigated sex differences in neutrophil stimulation and recruitment in ischemia/reperfusion (I/R; mesenteric or renal) or carrageenan pleurisy in rats or mice, as well as skin injury in human volunteers. The induction of potent chemoattractive mediators (chemokines) and neutrophil adhesion molecules were measured by real-time PCR, flow cytometry, and protein assays.ResultsMesenteric I/R in age-matched Wistar rats resulted in substantially more neutrophil accumulation and tissue injury at 2 h reperfusion in males than females. Using intravital microscopy, we show that the immediate (<30 min) neutrophil response to I/R is similar in males and females but that prolonged neutrophil recruitment occurs in males at sites local and distal to inflammatory insult partly due to an increase in circulating neutrophil populations with elevated surface expression of adhesion molecules. Sex differences in neutrophil kinetics were correlated with sustained induction of chemokine Cxcl5 in the tissue, circulation, and bone marrow of males but not females. Furthermore, blockade of Cxcl5 in males prior to ischemia resulted in neutrophil responses that were similar in magnitude to those in females. Conversely, administration of Cxcl5 to males in the absence of I/R was sufficient to increase levels of systemic neutrophils. Cxcl5 treatment of bone marrow neutrophils in vitro caused substantial induction of neutrophil-mobilizing cytokine granulocyte colony-stimulating factor (GCSF) and expression of β2 integrin that accounts for sexual dimorphism in circulating neutrophil populations in I/R. Moreover, male Cxcl5-stimulated bone marrow neutrophils had an increased capacity to adhere to β2 integrin ligand ICAM-1, implicating a greater sensitivity of male leukocytes to Cxcl5-mediated activation. Differential induction of Cxcl5 (human CXCL6) between the sexes was also evident in murine renal I/R, rat pleurisy, and human skin blisters and correlated with the magnitude of neutrophil accumulation in tissues.ConclusionsOur study reveals that sex-specific induction of chemokine Cxcl5/CXCL6 contributes to sexual dimorphism in neutrophil recruitment in diverse acute inflammatory responses partly due to increased stimulation and trafficking of bone marrow neutrophils in males.
| S-EPMC4661984 | biostudies-literature
Project description:Bam32 (B cell adaptor molecule of 32 kDa) functions in the immune responses of various leukocytes. However, the role of neutrophil Bam32 in inflammation is entirely unknown. Here, we determined the role of Bam32 in chemokine CXCL2-induced neutrophil chemotaxis in three mouse models of neutrophil recruitment. By using intravital microscopy in the mouse cremaster muscle, we found that transmigrated neutrophil number, neutrophil chemotaxis velocity, and total neutrophil chemotaxis distance were increased in Bam32-/- mice when compared with wild-type (WT) mice. In CXCL2-induced mouse peritonitis, the total emigrated neutrophils were increased in Bam32-/- mice at 2 but not 4 h. The CXCL2-induced chemotaxis distance and migration velocity of isolated Bam32-/- neutrophils in vitro were increased. We examined the activation of small GTPases Rac1, Rac2, and Rap1; the levels of phospho-Akt2 and total Akt2; and their crosstalk with Bam32 in neutrophils. The deficiency of Bam32 suppressed Rap1 activation without changing the activation of Rac1 and Rac2. The pharmacological inhibition of Rap1 by geranylgeranyltransferase I inhibitor (GGTI298) increased WT neutrophil chemotaxis. In addition, the deficiency of Bam32, as well as the inhibition of Rap1 activation, increased the levels of CXCL2-induced Akt1/2 phosphorylation at Thr308/309 in neutrophils. The inhibition of Akt by SH-5 attenuated CXCL2-induced adhesion and emigration in Bam32-/- mice. Together, our results reveal that Bam32 has a suppressive role in chemokine-induced neutrophil chemotaxis by regulating Rap1 activation and that this role of Bam32 in chemokine-induced neutrophil recruitment relies on the activation of PI3K effector Akt.
| S-EPMC7918626 | biostudies-literature
Project description:Hepatic microRNA was measured by Exiqon array in the mouse RRV model of biliary atresia miRNA expression profiling. For the miRNA microarray experiment, RRV and saline injected animals were euthanized at 3, 8, and 14 days post infection (n=5 per treatment, per time point), and total RNA was purified from liver using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. A pooled common control was assembled from an equal RNA mass from each of the samples, and samples and control were separately dye-labeled, and hybridized to a miRcury v11.0 miRNA microarray (Exiqon) at the University of Pennsylvania School of Medicine Microarray core facility. Sample hybridization intensities were scored relative to the common control, and raw intensity data were normalized and analyzed using the SAM add-in for Microsoft Excel. MiRNAs exhibiting a fold-change of greater than 10% up or down, at a false discovery rate of 5% were chosen for further study.
2011-11-04 | GSE33418 | GEO
Project description:Hepatic microRNA was measured by Exiqon array in the mouse RRV model of biliary atresia miRNA expression profiling. For the miRNA microarray experiment, RRV and saline injected animals were euthanized at 3, 8, and 14 days post infection (n=5 per treatment, per time point), and total RNA was purified from liver using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. A pooled common control was assembled from an equal RNA mass from each of the samples, and samples and control were separately dye-labeled, and hybridized to a miRcury v11.0 miRNA microarray (Exiqon) at the University of Pennsylvania School of Medicine Microarray core facility. Sample hybridization intensities were scored relative to the common control, and raw intensity data were normalized and analyzed using the SAM add-in for Microsoft Excel. MiRNAs exhibiting a fold-change of greater than 10% up or down, at a false discovery rate of 5% were chosen for further study. Three time points (3, 8, 14 days) x 2 conditions x 6 replicates - 1 failed sample = 35 samples
2011-11-04 | E-GEOD-33418 | biostudies-arrayexpress
Project description:The treatment of biliary atresia (BA) is predominantly surgical with firstly an attempt at restoration of bile flow from the native liver by wide excision of the obstructed, obliterated extrahepatic biliary tree to the level of the porta hepatis and a portoenterostomy using a long Roux loop-Kasai portoenterostomy (KPE). Liver transplantation is reserved for those that fail this and for those where surgery is considered futile for reasons of age or stage of disease. As the aetiology of BA remains ill-defined, so adjuvant treatment has been largely based on pragmatism, trial and error. Systematic analysis of the few randomized placebo-controlled trial data and less well-controlled cohort studies have suggested benefit from post-operative high-dose steroids and ursodeoxycholic acid (UDCA) while the benefit of long-term prophylactic antibiotics, bile acid sequestrants (e.g., colestyramine) or probiotics remains unproven. Newer modalities such as antiviral therapy (AVT), immunoglobulin, FXR agonists (e.g., obeticholic acid), ileal bile acid transporter (IBAT) antagonists (e.g., maralixibat) remain unproven. This article reviews the current evidence for the efficacy of adjuvant medical therapy in BA.
| S-EPMC7347763 | biostudies-literature
Project description:In patients with biliary atresia (BA), the extent of intrahepatic biliary fibrosis negatively correlates with successful surgical bypass of the congenital cholangiopathy as well as subsequent transplant-free survival. We recently linked the expansion of a population of prominin-1 (Prom1)-expressing hepatic progenitor cells to biliary fibrogenesis. Herein, we hypothesized that Prom1-expressing progenitor cells play a role in BA-associated fibrosis. Rhesus rotavirus (RRV)-mediated experimental BA was induced in newborn mice homozygous for the transgene Prom1cre-ert2-nlacz , which was knocked in to the Prom1 gene locus, thus creating functional Prom1 knockout (KO) mice, and their wildtype (WT) littermates. Clinical data and tissue samples from BA infants from the Childhood Liver Disease Research Consortium were analyzed. Extrahepatic biliary obliteration was present in both WT and KO mice; there was no difference in serum total bilirubin (TBili) levels. The intrahepatic periportal expansion of the PROM1pos cell population, typically observed in RRV-induced BA, was absent in KO mice. RRV-treated KO mice demonstrated significantly fewer cytokeratin-19 (CK19)-positive ductular reactions (P = 0.0004) and significantly less periportal collagen deposition (P = 0.0001) compared with WT. RRV-treated KO mice expressed significantly less integrin-β6, which encodes a key biliary-specific subunit of a transforming growth factor (TGF) β activator (P = 0.0004). Infants with successful biliary drainage (Tbili ≤1.5 mg/dL within 3 months postoperatively), which is highly predictive of increased transplant-free survival, expressed significantly less hepatic PROM1, CK19, and COLLAGEN-1α compared with those with TBili >1.5 (P < 0.05). Conclusion: Prom1 plays an important role in biliary fibrogenesis, in part through integrin-mediated TGF pathway activation.
| S-EPMC6541523 | biostudies-literature
Project description:We examined gene regulation in murine lungs after hind-limb vessel occlusion and reperfusion. A rapid increase of transcript for the AMP deaminase 3 gene (AMPD3) and its enzymatic activity (EC) generating inosine monophosphate (IMP) were identified with transcripts located in bronchial and alveolar epithelium. AMP deaminase inhibitor decreased IMP levels and significantly enhanced neutrophil recruitment within lung tissue during reperfusion. In addition, IMP inhibited cytokine-initiated neutrophil infiltration in vivo and selectively attenuated neutrophil rolling by 90% in microvessels. We prepared labeled IMP and demonstrated that IMP specifically binds to neutrophils. IMP also stimulated binding of gamma-[(35)S]thio-GTP, suggesting that IMP is a potent regulator of neutrophils. Taken together, these results elucidate a previously unrecognized mechanism that protects tissues from the potentially deleterious consequences of aberrant neutrophil accumulation. Moreover, they are relevant for new therapeutic approaches to regulate neutrophil responses in inflammation and vascular disease.
| S-EPMC18224 | biostudies-literature
Project description:BackgroundBiliary atresia (BA) is a severe hepatobiliary disease in infants that ultimately results in hepatic failure; however, its pathological mechanism is poorly elucidated. Current surgical options, including Kasai hepatoportoenterostomy and orthotopic liver organ transplantations, are palliative; thus, innovation in BA therapy is urgent.MethodsTo examine whether BA-specific post-natal stem cells are feasible for autologous cell source for BA treatment, we isolated from human exfoliated deciduous teeth, namely BA-SHED, using a standard colony-forming unit fibroblast (CFU-F) method and compared characteristics as mesenchymal stem cells (MSCs) to healthy donor-derived control SHED, Cont-SHED. BA-SHED and Cont-SHED were intrasplenically transplanted into chronic carbon tetrachloride (CCl4)-induced liver fibrosis model mice, followed by the analysis of bile drainage function and donor integration in vivo. Immunohistochemical assay was examined for the regeneration of intrahepatic bile ducts in the recipient's liver using anti-human specific keratin 19 (KRT19) antibody.ResultsBA-SHED formed CFU-F, expressed MSC surface markers, and exhibited in vitro mesenchymal multipotency similar to Cont-SHED. BA-SHED showed less in vitro hepatogenic potency than Cont-SHED. Cont-SHED represented in vivo bile drainage function and KRT19-positive biliary regeneration in chronic carbon tetrachloride-induced liver fibrosis model mice. BA-SHED failed to show in vivo biliary potency and bile drainage function compared to Cont-SHED.ConclusionThese findings indicate that BA-SHED are not feasible source for BA treatment, because BA-SHED may epigenetically modify the underlying prenatal and perinatal BA environments. In conclusion, these findings suggest that BA-SHED-based studies may provide a platform for understanding the underlying molecular mechanisms of BA development and innovative novel modalities in BA research and treatment.
| S-EPMC8607730 | biostudies-literature
Project description:Biliary atresia (BA) is a progressive fibro-inflammatory disorder that is the leading indication for liver transplantation in children. Although there is evidence implicating genetic, infectious, environmental, and inflammatory causes, the etiology of BA remains unknown. We have recently reported that cholangiocytes from BA patients showed decreased DNA methylation relative to disease- and non-disease controls, supporting a potential role for DNA hypomethylation in BA etiopathogenesis. In the current study, we examined the methylation status of specific genes in human BA livers using methylation microarray technology. We found global DNA hypomethylation in BA samples as compared to disease- and non-disease controls at specific genetic loci. Hedgehog pathway members, SHH and GLI2, known to be upregulated in BA, were both hypomethylated, validating this approach as an investigative tool. Another region near the PDGFA locus was the most significantly hypomethylated in BA, suggesting potential aberrant expression. Validation assays confirmed increased transcriptional and protein expression of PDGFA in BA livers. We also show that PDGF-A protein is specifically localized to cholangiocytes in human liver samples. Injection of PDGF-AA protein dimer into zebrafish larvae caused biliary developmental and functional defects. In addition, activation of the Hedgehog pathway caused increased expression of PDGF-A in zebrafish larvae, providing a previously unrecognized link between PDGF and the Hedgehog pathway. Our findings implicate DNA hypomethylation as a specific factor in mediating overexpression of genes associated with BA and identify PDGF as a new candidate in BA pathogenesis.
| S-EPMC4806872 | biostudies-literature
Project description:Biliary atresia (BA) is notable for marked ductular reaction and rapid development of fibrosis. Activation of the Hedgehog (Hh) pathway promotes the expansion of populations of immature epithelial cells that coexpress mesenchymal markers and may be profibrogenic. We examined the hypothesis that in BA excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Livers and remnant extrahepatic ducts from BA patients were evaluated by quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and immunostaining for Hh ligands, target genes, and markers of mesenchymal cells or ductular progenitors. Findings were compared to children with genetic cholestatic disease, age-matched deceased donor controls, and adult controls. Ductular cells isolated from adult rats with and without bile duct ligation were incubated with Hh ligand-enriched medium ± Hh-neutralizing antibody to determine direct effects of Hh ligands on epithelial to mesenchymal transition (EMT) marker expression. Livers from pediatric controls showed greater innate Hh activation than adult controls. In children with BA, both intra- and extrahepatic ductular cells demonstrated striking up-regulation of Hh ligand production and increased expression of Hh target genes. Excessive accumulation of Hh-producing cells and Hh-responsive cells also occurred in other infantile cholestatic diseases. Further analysis of the BA samples demonstrated that immature ductular cells with a mesenchymal phenotype were Hh-responsive. Treating immature ductular cells with Hh ligand-enriched medium induced mesenchymal genes; neutralizing Hh ligands inhibited this.BA is characterized by excessive Hh pathway activity, which stimulates biliary EMT and may contribute to biliary dysmorphogenesis. Other cholestatic diseases show similar activation, suggesting that this is a common response to cholestatic injury in infancy.
| S-EPMC3074103 | biostudies-literature