RESUMEN
Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.
Asunto(s)
Atresia Biliar , Proteínas de la Cápside , Modelos Animales de Enfermedad , Endocitosis , Infecciones por Rotavirus , Rotavirus , Atresia Biliar/metabolismo , Atresia Biliar/virología , Animales , Ratones , Infecciones por Rotavirus/metabolismo , Infecciones por Rotavirus/virología , Humanos , Proteínas de la Cápside/metabolismo , Receptor Toll-Like 3/metabolismo , Sitios de Unión , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSC70/genética , Ratones Noqueados , FN-kappa B/metabolismo , Transducción de Señal , Clatrina/metabolismo , Ratones Endogámicos C57BL , Quimiocina CXCL10RESUMEN
BACKGROUND AND AIMS: Biliary atresia (BA) is a devastating cholangiopathy of infancy. Upon diagnosis, surgical reconstruction by Kasai hepatoportoenterostomy (HPE) restores biliary drainage in a subset of patients, but most patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling that of human BA. High-mobility group box 1 (HMGB1) is an important member of the danger-associated molecular patterns capable of mediating inflammation during infection-associated responses. In this study, we investigated the role of HMGB1 in BA pathogenesis. APPROACH AND RESULTS: In cholangiocytes, RRV induced the expression and release of HMGB1 through the p38 mitogen-activated protein kinase signaling pathway, and inhibition of p38 blocked HMGB1 release. Treatment of cholangiocytes with ethyl pyruvate suppressed the release of HMGB1. Administration of glycyrrhizin in vivo decreased symptoms and increased survival in the murine model of BA. HMGB1 levels were measured in serum obtained from infants with BA enrolled in the PROBE and START studies conducted by the Childhood Liver Disease Research Network. High HMGB1 levels were found in a subset of patients at the time of HPE. These patients had higher bilirubin levels 3 months post-HPE and a lower survival of their native liver at 2 years. CONCLUSIONS: These results suggest that HMGB1 plays a role in virus induced BA pathogenesis and could be a target for therapeutic interventions in a subset of patients with BA and high HMGB1.
Asunto(s)
Atresia Biliar/patología , Enfermedad Hepática en Estado Terminal/epidemiología , Proteína HMGB1/sangre , Proteína HMGB1/metabolismo , Infecciones por Rotavirus/patología , Animales , Animales Recién Nacidos , Conductos Biliares/metabolismo , Conductos Biliares/patología , Conductos Biliares/cirugía , Atresia Biliar/sangre , Atresia Biliar/cirugía , Atresia Biliar/virología , Bilirrubina/sangre , Biomarcadores/sangre , Línea Celular , Preescolar , Chlorocebus aethiops , Modelos Animales de Enfermedad , Enfermedad Hepática en Estado Terminal/patología , Células Epiteliales , Humanos , Lactante , Recién Nacido , Ratones , Portoenterostomía Hepática , Medición de Riesgo , Factores de Riesgo , Rotavirus/metabolismo , Rotavirus/patogenicidad , Infecciones por Rotavirus/virología , Resultado del TratamientoRESUMEN
BACKGROUND AND AIMS: Biliary atresia (BA) is a devastating neonatal cholangiopathy that progresses to fibrosis and end-stage liver disease by 2 years of age. Portoenterostomy may reestablish biliary drainage, but, despite drainage, virtually all afflicted patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival. APPROACH AND RESULTS: In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling human BA and has been used to study mechanistic aspects of the disease. Unfortunately, nearly all RRV-infected pups succumb by day of life 14. Thus, in this study we generated an RRV-TUCH rotavirus reassortant (designated as TR(VP2,VP4) ) that when injected into newborn mice causes an obstructive jaundice phenotype with lower mortality rates. Of the mice that survived, 63% developed Ishak stage 3-5 fibrosis with histopathological signs of inflammation/fibrosis and bile duct obstruction. CONCLUSIONS: This model of rotavirus-induced neonatal fibrosis will provide an opportunity to study disease pathogenesis and has potential to be used in preclinical studies with an objective to identify therapeutic targets that may alter the course of BA.
Asunto(s)
Atresia Biliar/complicaciones , Modelos Animales de Enfermedad , Cirrosis Hepática/virología , Ratones , Virus Reordenados , Rotavirus , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Ictericia Obstructiva/virología , Cirrosis Hepática/etiología , Ratones Endogámicos BALB CRESUMEN
Vertical sleeve gastrectomy (VSG) is the best current therapy for remission of obesity and its co-morbidities. It is understood to alter the enterohepatic circulation of bile acids in vivo. Fibroblast growth factor 19 (FGF19) in human and its murine orthologue Fgf15 plays a pivotal role in this bile acid driven enterohepatic signaling. The present study evaluated the metabolic outcomes of VSG in Fgf15 deficient mice. 6-8 weeks old male wildtype mice (WT) and Fgf15 deficient mice (KO) were fed a high fat diet (HFD) for 8 weeks. At 8th week of diet, both WT and KO mice were randomly distributed to VSG or sham surgery. Post-surgery, mice were observed for 8 weeks while fed a HFD and then euthanized to collect tissues for experimental analysis. Fgf15 deficient (KO) mice lost weight post VSG, but glucose tolerance in KO mice did not improve post VSG compared to WT mice. Enteroids derived from WT and KO mice proliferated with bile acid exposure in vitro. Post VSG both WT and KO mice had similarly altered bile acid enterohepatic flux, however Fgf15 deficient mice post VSG had increased hepatic accumulation of free and esterified cholesterol leading to lipotoxicity related ER stress, inflammasome activation, and increased Fgf21 expression. Intact Fgf15 mediated enterohepatic bile acid signaling, but not changes in bile acid flux, appear to be important for the metabolic improvements post-murine bariatric surgery. These novel data introduce a potential point of distinction between bile acids acting as ligands compared to their canonical downstream signaling pathways.
RESUMEN
Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice.IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.
Asunto(s)
Atresia Biliar/patología , Proteínas de la Cápside/genética , Proteínas Mutantes/genética , Mutación Puntual , Rotavirus/patogenicidad , Animales , Animales Recién Nacidos , Atresia Biliar/virología , Proteínas de la Cápside/metabolismo , Modelos Animales de Enfermedad , Ratones , Proteínas Mutantes/metabolismo , Genética Inversa , Rotavirus/genética , Tropismo Viral , Replicación ViralRESUMEN
Biliary atresia (BA) is a neonatal inflammatory cholangiopathy that requires surgical intervention by Kasai portoenterostomy to restore biliary drainage. Even with successful portoenterostomy, most patients diagnosed with BA progress to end-stage liver disease, necessitating a liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of neonatal mice induces an inflammatory obstructive cholangiopathy that parallels human BA. The model is triggered by RRV viral protein (VP)4 binding to cholangiocyte cell-surface proteins. High mobility group box 1 (HMGB1) protein is a danger-associated molecular pattern that when released extracellularly moderates innate and adaptive immune response. In this study, we investigated how mutations in three RRV VP4-binding sites, RRVVP4-K187R (sialic acid-binding site), RRVVP4-D308A (integrin α2ß1-binding site), and RRVVP4-R446G (heat shock cognate 70 [Hsc70]-binding site), affects infection, HMGB1 release, and the murine model of BA. Newborn pups injected with RRVVP4-K187R and RRVVP4-D308A developed an obstruction within the extrahepatic bile duct similar to wild-type RRV, while those infected with RRVVP4-R446G remained patent. Infection with RRVVP4-R446G induced a lower level of HMGB1 release from cholangiocytes and in the serum of infected pups. RRV infection of HeLa cells lacking Hsc70 resulted in no HMGB1 release, while transfection with wild-type Hsc70 into HeLa Hsc70-deficient cells reestablished HMGB1 release, indicating a mechanistic role for Hsc70 in its release. Conclusion: Binding to Hsc70 contributes to HMGB1 release; therefore, Hsc70 potentially serves as a therapeutic target for BA.
Asunto(s)
Atresia Biliar , Infecciones por Rotavirus , Rotavirus , Animales , Animales Recién Nacidos , Atresia Biliar/etiología , Sitios de Unión , Modelos Animales de Enfermedad , Células HeLa , Humanos , Integrina alfa2beta1 , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Ácido N-Acetilneuramínico , Rotavirus/genética , Infecciones por Rotavirus/metabolismo , Proteínas ViralesRESUMEN
Collagen XXII (COL22A1) is a quantitatively minor collagen, which belongs to the family of fibril-associated collagens with interrupted triple helices. Its biological function has been poorly understood. Here, we used a genome-editing approach to generate a loss-of-function mutant in zebrafish col22a1 Homozygous mutant adults exhibit increased incidence of intracranial hemorrhages, which become more prominent with age and after cardiovascular stress. Homozygous col22a1 mutant embryos show higher sensitivity to cardiovascular stress and increased vascular permeability, resulting in a greater percentage of embryos with intracranial hemorrhages. Mutant embryos also exhibit dilations and irregular structure of cranial vessels. To test whether COL22A1 is associated with vascular disease in humans, we analyzed data from a previous study that performed whole-exome sequencing of 45 individuals from seven families with intracranial aneurysms. The rs142175725 single-nucleotide polymorphism was identified, which segregated with the phenotype in all four affected individuals in one of the families, and affects a highly conserved E736 residue in COL22A1 protein, resulting in E736D substitution. Overexpression of human wild-type COL22A1, but not the E736D variant, partially rescued the col22a1 loss-of-function mutant phenotype in zebrafish embryos. Our data further suggest that the E736D mutation interferes with COL22A1 protein secretion, potentially leading to endoplasmic reticulum stress. Altogether, these results argue that COL22A1 is required to maintain vascular integrity. These data further suggest that mutations in COL22A1 could be one of the risk factors for intracranial aneurysms in humans.
Asunto(s)
Vasos Sanguíneos/patología , Colágeno/genética , Aneurisma Intracraneal/genética , Mutación/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Secuencia de Bases , Colágeno/metabolismo , Embrión no Mamífero/metabolismo , Estrés del Retículo Endoplásmico , Endotelio Vascular/patología , Endotelio Vascular/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/patología , Gastrulación , Eliminación de Gen , Hemorragia/patología , Homocigoto , Humanos , Aneurisma Intracraneal/patología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Temperatura , Regulación hacia Arriba/genética , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismoRESUMEN
OBJECTIVE: Vertical sleeve gastrectomy (VSG) results in weight loss and increased bile acids (BA) and fibroblast growth factor 19 (FGF19) levels. FGF21 shares essential cofactors with FGF19, but its physiology early post-VSG has not been assessed. METHODS: Ten adolescents (17.4 ± 0.5 years and BMI 51.5 ± 2.5 kg/m2 ) were enrolled. Fasting and postmeal (100 mL Ensure™) samples (0-120 min) were collected (pre-VSG [V1], 1 [V2], and 3 months [V3] post-VSG) for analysis of BA, FGF19, and FGF21. RESULTS: Post-VSG subjects lost weight (V2 11.8 ± 0.8 kg; V3 21.9 ± 1.7 kg). BA and FGF19 increased by V2, 143.6% at 30 min and 74.9% at 90 min post-meal, respectively. BA hydrophobicity index also improved by V3, 21.1% at 30 min post-meal. Interestingly, fasting and 120-min post-meal FGF21 levels at V2 were increased by 135.7% and 253.9%, respectively, but then returned to baseline at V3. BA levels correlated with FGF21 at V2 (P = 0.003, r = 0.89), and body weight lost post-VSG correlated with FGF21 levels (V2; P = 0.012, R = 0.82). CONCLUSIONS: Expected changes were seen in BA and FGF19 biology after VSG in adolescents, but novel changes were seen in correlation between the early postsurgical increase in FGF21 and weight loss, suggesting that FGF21 may play a role in energy balance postoperatively, and further investigation is warranted.