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Depletion of Microrna-21 Reduces Infiltration of Macrophages and Neutrophils in the Liver and Attenuates Inflammatory Cytokine Production in Liver Macrophages During Experimental Cholestatic Liver Injury
stellate cells (Collagen 1A1, TGFβ1 and Synaptophysin 9). Results: Exposure to GWIrelated chemicals alone had no effects on liver damage, intrahepatic biliary mass, inflammatory cytokine expression, hepatic stellate cell activation or fibrosis when measured 10 weeks after the last GWI exposure. However, GWI-related chemicals caused increased liver damage, IBDM and cholangiocyte proliferation after BDL compared to naïve rats undergoing BDL surgery. Furthermore there was increased expression of IL1β and TNFα after BDL in GWI rats compared to naïve rats, suggesting an enhanced inflammatory response. Lastly, there was increased Synaptophysin 9 expression and a greater fibrotic response in rats exposed to GWI-related chemicals compared to naïve rats. Conclusion: While exposure to GWIrelated chemicals had no lasting effect on the parameters of liver damage when assessed 10 weeks after the cessation of exposure, there was an enhanced response to BDL surgery when compared to naïve animals. Taken together, these data suggest that GWI patients could be predisposed to developing worse liver pathology in response to cholestatic liver injury when compared to patients without GWI.
575 DEPLETION OF MICRORNA-21 REDUCES INFILTRATION OF MACROPHAGES AND NEUTROPHILS IN THE LIVER AND ATTENUATES INFLAMMATORY CYTOKINE PRODUCTION IN LIVER MACROPHAGES DURING EXPERIMENTAL CHOLESTATIC LIVER INJURY Keisaku Sato, Lindsey Kennedy, Tianhao Zhou, Kelly M. McDaniel, Sugeily RamosLorenzo, Ying Wan, Julie Venter, Heather L. Francis, Shannon Glaser, Gianfranco Alpini, Fanyin Meng
AASLD Abstracts
573
Background: Kupffer cells are liver-resident macrophages that play an important role in cholestatic liver diseases via cytokine production and recruitment of other cells such as neutrophils. MicroRNA-21 (miR-21) is a key microRNA in liver disorders such as liver cancer and cholestatic liver injury. Although the detailed functions of miR-21 in cholestatic liver diseases are still unclear, our recent study has shown that miR-21 knockout mice have ameliorated liver fibrosis following bile duct ligation (BDL) surgery. In this current study, we sought functional roles of miR-21 in cytokine production and infiltration of inflammatory cells during cholestatic liver inflammation. Methods: Wild-type (WT) and miR-21 knockout mice were subjected to sham or BDL. After one week, mice were sacrificed and liver tissues were collected. Frozen liver tissue sections were cut with 6 µm thickness and placed on membrane slides. After liver tissues were fixed in 100% ice-cold acetone, liver macrophages were stained by anti-F4/80 primary and Cy2-conjugated secondary antibodies. Stained macrophages were cut and collected by laser capture microdissection using Leica LMD7. RNAs were extracted from isolated pooled macrophages and RT-qPCR was performed to analyze cytokine production for inflammation (IL-1β, IL-6, TNFα and CCL2). Frozen liver sections on glass slides were also fixed in paraformaldehyde and permeabilized by 1% Triton X-100. Kupffer cells and infiltrated bone marrow-derived macrophages were stained by antiF4/80 primary and Cy3-conjugated secondary antibodies. Infiltrated neutrophils were also stained by anti-myeloperoxidase primary antibody. Stained cells were observed under fluorescent microscope with DAPI as a counterstain. Results: miR-21 knockout mice show attenuated liver damage and fibrosis after BDL compared with WT. Immunofluorescence showed increased infiltration of macrophages and neutrophils in the liver following BDL, but BDL miR-21 knockout mice showed reduced macrophage and neutrophil infiltration compared with BDL WT. Laser capture microdissection followed by RT-qPCR showed that WT macrophages produced enhanced levels of all cytokines analyzed after BDL leading to liver inflammation. Macrophages with miR-21 deficiency, however, did not show increased expression levels for any cytokines analyzed after BDL. Conclusion: The present study suggests that miR-21 is associated with cytokine production in liver macrophages as well as infiltration of Kupffer cells, bone marrow-derived macrophages and neutrophils. As a result, insufficient numbers of infiltrated cells and reduced levels of inflammatory cytokines in the liver attenuated liver inflammation in miR-21 knockout mice during BDL. These findings present a potential role of miR-21 as a novel therapeutic target for cholestatic liver inflammation.
WHOLE EXOME SEQUENCING (WES) IDENTIFIES CILIOPATHY AND LATERALITY CANDIDATE GENES FOR BILIARY ATRESIA (BA) John-Paul Berauer, Anya Mezina, Aniko Sabo, Madhuri Hegde, David H. Perlmutter, Saul J. Karpen Background: BA is a severe cholangiopathy of infancy in which progressive fibro-obliteration of the extra- and intrahepatic bile ducts leads to cholestatic liver disease, and, ultimately, biliary cirrhosis. Among children, BA is the most common cause of end-stage liver disease and the primary indication for liver transplantation, yet underlying etiologies remain unknown. In ~5-10% of infants affected with BA, major congenital anomalies and laterality defects coexist along with biliary tract dysgenesis, suggesting a role for genetic contributions to the malformation of these structures. We hypothesized that the application of WES to this unique subset of BA patients, herein referred to as BA+L (L:laterality), would lead to the discovery of etiologic candidate genes for BA. Methods: We sequenced the exomes of 20 BA+L subjects and their parents (trios). Subjects were identified from the NIH-supported Childhood Liver Disease Research and Education Network (ChiLDReN, childrennetwork.org) based on the availability of DNA specimens in the ChiLDReN biorepository and the presence of at least one laterality defect (e.g., splenic abnormalities, intestinal malrotation, abdominal heterotaxy, vascular abnormalities, and heart disease) or cystic renal dysplasia. Exomes from each trio were enriched using NimbleGen SeqCap technology and underwent systematic analyses by standard variant analysis software with comparison to a reference genome. Detailed examinations of each trio focused on a ciliopathy/laterality gene set and putative BA susceptibility loci. Candidate gene prioritization methods included the use of computational protein function prediction algorithms (SIFT, PolyPhen-2, and CADD) and selection of variants with a minor allele frequency <1%. Results: Unbiased variant analyses did not reveal a single gene with pathogenic alterations common to the 20 BA+L subjects. However, a focused exploration of variants in genes known to participate in laterality defects and ciliopathies led to the identification of rare and potentially deleterious variants in 15 candidate genes in 12 of the 20 BA+L subjects (6 were compound heterozygotes). Several of these candidate genes have been previously associated with BA (CFC1, HNF1B, ZIC3) while others (ACVR2B, BCOR, DNAH11, HSPG2, HYDIN, IFT172, LRP2, PKD1, RPGR, SHROOM3, TBX5, ZFPM2) were newly associated with BA. Conclusion: These results underscore the utility of WES in the identification of candidate genes for genetically complex and heterogeneous disorders such as BA, expand the genetic spectrum of this rare disorder, and lend credence to the theory that ciliary dysgenesis or dysfunction contributes to the etiopathogenesis of BA in select patients. Cell and animal-based validation studies are needed to investigate the role of these promising candidate genes as susceptibility factors for BA.
576 BODY MASS INDEX IS NOT ASSOCIATED WITH THE RISK OF LIVER BIOPSY-RELATED COMPLICATIONS IN CHILDREN Alice Y. Wang, Peter Church, Benson Law, Simon Ling, Philip John, Marialena Mouzaki
574 Background/Hypothesis Little is known about the effect of body habitus on the risk of complications after liver biopsy. We hypothesized that liver biopsy-related complications are more common in children with higher BMI, and that the risk of complications is higher when liver biopsies are performed to confirm the diagnosis of non-alcoholic fatty liver disease (NAFLD). Methods In this retrospective, single center study, the medical records of all children, ages 2-19 years, who had image-guided, percutaneous liver biopsies performed by interventional radiologists at the Hospital for Sick Children were reviewed. Data extracted included demographics, anthropometrics, biopsy indication, needle size, number of passes, and laboratory values (hemoglobin, platelets, and INR) pre- and post-biopsy. Complications were categorized using the Society of Interventional Radiology classification system. Association between complications and covariates of interest was sought using univariable and then multivariable logistic regression. Results The charts of 700 children (53% male, median age 12 [IQR 7-15] years) were reviewed. The most common indication for liver biopsy was to investigate the cause of abnormal liver biochemistry (42%) with 5% of biopsies confirming a diagnosis of NAFLD. There were 44 minor complications (prevalence 6%): pain (n=37), cutaneous hematoma (n=3, 1 of which developed after 24 hours), fever (n=3), vomiting (n= 2), and 5 major complications in 5 different patients (prevalence 0.4%): bleed requiring blood products (n=1), sepsis (n=2), hospitalization (n=2, one within 24 hours because of a hematoma and one after 24h because of fever). Two of the 3 patients who had bleeding complications post biopsy had normal platelets (>150 x109/L) and INR (≤1.1) prior to the procedure; the third patient was in acute liver failure at the time of biopsy (INR 1.8, PLT 152 x109/L). Significant predictors of post-biopsy complications found on univariable analysis included lower height and weight z-scores, and a trend towards increasing age (Table). After controlling for confounders, such as age, BMI z-score, and needle gauge, a diagnosis of NAFLD was associated with a 3-fold decreased odds of complications (OR=0.23, p=0.05)
CHOLESTATIC LIVER INJURY IS EXACERBATED BY PRIOR EXPOSURE TO GULF WAR ILLNESS-RELATED CHEMICALS Matthew McMillin, Stephanie Grant, Gabriel Frampton, Maheedhar Kodali, Ashok Shetty, Sharon DeMorrow Gulf war illness (GWI) is a term used to describe a chronic multisymptom disorder affecting military veterans of the 1990-91 Persian Gulf War. Such symptoms include chronic fatigue, muscle pain, rashes and cognitive problems. GWI has been linked to exposure to a combination of chemicals including the anti-nerve gas drug pyridostigmine bromide (PB) and pesticides (eg DEET or permethrin) with a combination of all of these chemicals likely increasing pathogenesis. While no evidence exists that exposure to GWI-related chemicals causes any particular liver pathology, there is circumstantial evidence to suggest that exposure may reduce the ability of the liver to cope and repair itself after chronic liver injury. The aim of this study is to assess the effects of GWI-related chemicals on cholangiocyte proliferation and liver fibrosis after bile duct ligation (BDL). Methods: Male Sprague Dawley rats were treated with PB (2mg/kg/day; gavage), DEET (60 mg/kg/day, dermal application) and Permethrin (0.2 mg/kg/day) followed by manual restraint stress (15 minutes) daily for 28 consecutive days. Ten weeks after the last treatment, GWI rats or naïve age-matched controls underwent BDL (or sham) surgeries. Cholangiocyte proliferation and intrahepatic bile duct mass (IBDM) was assessed in vivo by immunohistochemistry and qPCR using proliferating cellular nuclear antigen (PCNA) or cytokeratin 19 (CK19) antibodies respectively. Serum chemistry for AST, ALT, alkaline phosphatase and bilirubin were assessed. Proinflammatory cytokine expression was assessed by qPCR and EIA. Liver fibrosis was measured by Sirius red staining and qPCR in total liver extracts for markers of fibrosis and activated hepatic
AASLD Abstracts
S-1064
575 DEPLETION OF MICRORNA-21 REDUCES INFILTRATION OF MACROPHAGES AND NEUTROPHILS IN THE LIVER AND ATTENUATES INFLAMMATORY CYTOKINE PRODUCTION IN LIVER MACROPHAGES DURING EXPERIMENTAL CHOLESTATIC LIVER INJURY Keisaku Sato, Lindsey Kennedy, Tianhao Zhou, Kelly M. McDaniel, Sugeily RamosLorenzo, Ying Wan, Julie Venter, Heather L. Francis, Shannon Glaser, Gianfranco Alpini, Fanyin Meng
AASLD Abstracts
573
Background: Kupffer cells are liver-resident macrophages that play an important role in cholestatic liver diseases via cytokine production and recruitment of other cells such as neutrophils. MicroRNA-21 (miR-21) is a key microRNA in liver disorders such as liver cancer and cholestatic liver injury. Although the detailed functions of miR-21 in cholestatic liver diseases are still unclear, our recent study has shown that miR-21 knockout mice have ameliorated liver fibrosis following bile duct ligation (BDL) surgery. In this current study, we sought functional roles of miR-21 in cytokine production and infiltration of inflammatory cells during cholestatic liver inflammation. Methods: Wild-type (WT) and miR-21 knockout mice were subjected to sham or BDL. After one week, mice were sacrificed and liver tissues were collected. Frozen liver tissue sections were cut with 6 µm thickness and placed on membrane slides. After liver tissues were fixed in 100% ice-cold acetone, liver macrophages were stained by anti-F4/80 primary and Cy2-conjugated secondary antibodies. Stained macrophages were cut and collected by laser capture microdissection using Leica LMD7. RNAs were extracted from isolated pooled macrophages and RT-qPCR was performed to analyze cytokine production for inflammation (IL-1β, IL-6, TNFα and CCL2). Frozen liver sections on glass slides were also fixed in paraformaldehyde and permeabilized by 1% Triton X-100. Kupffer cells and infiltrated bone marrow-derived macrophages were stained by antiF4/80 primary and Cy3-conjugated secondary antibodies. Infiltrated neutrophils were also stained by anti-myeloperoxidase primary antibody. Stained cells were observed under fluorescent microscope with DAPI as a counterstain. Results: miR-21 knockout mice show attenuated liver damage and fibrosis after BDL compared with WT. Immunofluorescence showed increased infiltration of macrophages and neutrophils in the liver following BDL, but BDL miR-21 knockout mice showed reduced macrophage and neutrophil infiltration compared with BDL WT. Laser capture microdissection followed by RT-qPCR showed that WT macrophages produced enhanced levels of all cytokines analyzed after BDL leading to liver inflammation. Macrophages with miR-21 deficiency, however, did not show increased expression levels for any cytokines analyzed after BDL. Conclusion: The present study suggests that miR-21 is associated with cytokine production in liver macrophages as well as infiltration of Kupffer cells, bone marrow-derived macrophages and neutrophils. As a result, insufficient numbers of infiltrated cells and reduced levels of inflammatory cytokines in the liver attenuated liver inflammation in miR-21 knockout mice during BDL. These findings present a potential role of miR-21 as a novel therapeutic target for cholestatic liver inflammation.
WHOLE EXOME SEQUENCING (WES) IDENTIFIES CILIOPATHY AND LATERALITY CANDIDATE GENES FOR BILIARY ATRESIA (BA) John-Paul Berauer, Anya Mezina, Aniko Sabo, Madhuri Hegde, David H. Perlmutter, Saul J. Karpen Background: BA is a severe cholangiopathy of infancy in which progressive fibro-obliteration of the extra- and intrahepatic bile ducts leads to cholestatic liver disease, and, ultimately, biliary cirrhosis. Among children, BA is the most common cause of end-stage liver disease and the primary indication for liver transplantation, yet underlying etiologies remain unknown. In ~5-10% of infants affected with BA, major congenital anomalies and laterality defects coexist along with biliary tract dysgenesis, suggesting a role for genetic contributions to the malformation of these structures. We hypothesized that the application of WES to this unique subset of BA patients, herein referred to as BA+L (L:laterality), would lead to the discovery of etiologic candidate genes for BA. Methods: We sequenced the exomes of 20 BA+L subjects and their parents (trios). Subjects were identified from the NIH-supported Childhood Liver Disease Research and Education Network (ChiLDReN, childrennetwork.org) based on the availability of DNA specimens in the ChiLDReN biorepository and the presence of at least one laterality defect (e.g., splenic abnormalities, intestinal malrotation, abdominal heterotaxy, vascular abnormalities, and heart disease) or cystic renal dysplasia. Exomes from each trio were enriched using NimbleGen SeqCap technology and underwent systematic analyses by standard variant analysis software with comparison to a reference genome. Detailed examinations of each trio focused on a ciliopathy/laterality gene set and putative BA susceptibility loci. Candidate gene prioritization methods included the use of computational protein function prediction algorithms (SIFT, PolyPhen-2, and CADD) and selection of variants with a minor allele frequency <1%. Results: Unbiased variant analyses did not reveal a single gene with pathogenic alterations common to the 20 BA+L subjects. However, a focused exploration of variants in genes known to participate in laterality defects and ciliopathies led to the identification of rare and potentially deleterious variants in 15 candidate genes in 12 of the 20 BA+L subjects (6 were compound heterozygotes). Several of these candidate genes have been previously associated with BA (CFC1, HNF1B, ZIC3) while others (ACVR2B, BCOR, DNAH11, HSPG2, HYDIN, IFT172, LRP2, PKD1, RPGR, SHROOM3, TBX5, ZFPM2) were newly associated with BA. Conclusion: These results underscore the utility of WES in the identification of candidate genes for genetically complex and heterogeneous disorders such as BA, expand the genetic spectrum of this rare disorder, and lend credence to the theory that ciliary dysgenesis or dysfunction contributes to the etiopathogenesis of BA in select patients. Cell and animal-based validation studies are needed to investigate the role of these promising candidate genes as susceptibility factors for BA.
576 BODY MASS INDEX IS NOT ASSOCIATED WITH THE RISK OF LIVER BIOPSY-RELATED COMPLICATIONS IN CHILDREN Alice Y. Wang, Peter Church, Benson Law, Simon Ling, Philip John, Marialena Mouzaki
574 Background/Hypothesis Little is known about the effect of body habitus on the risk of complications after liver biopsy. We hypothesized that liver biopsy-related complications are more common in children with higher BMI, and that the risk of complications is higher when liver biopsies are performed to confirm the diagnosis of non-alcoholic fatty liver disease (NAFLD). Methods In this retrospective, single center study, the medical records of all children, ages 2-19 years, who had image-guided, percutaneous liver biopsies performed by interventional radiologists at the Hospital for Sick Children were reviewed. Data extracted included demographics, anthropometrics, biopsy indication, needle size, number of passes, and laboratory values (hemoglobin, platelets, and INR) pre- and post-biopsy. Complications were categorized using the Society of Interventional Radiology classification system. Association between complications and covariates of interest was sought using univariable and then multivariable logistic regression. Results The charts of 700 children (53% male, median age 12 [IQR 7-15] years) were reviewed. The most common indication for liver biopsy was to investigate the cause of abnormal liver biochemistry (42%) with 5% of biopsies confirming a diagnosis of NAFLD. There were 44 minor complications (prevalence 6%): pain (n=37), cutaneous hematoma (n=3, 1 of which developed after 24 hours), fever (n=3), vomiting (n= 2), and 5 major complications in 5 different patients (prevalence 0.4%): bleed requiring blood products (n=1), sepsis (n=2), hospitalization (n=2, one within 24 hours because of a hematoma and one after 24h because of fever). Two of the 3 patients who had bleeding complications post biopsy had normal platelets (>150 x109/L) and INR (≤1.1) prior to the procedure; the third patient was in acute liver failure at the time of biopsy (INR 1.8, PLT 152 x109/L). Significant predictors of post-biopsy complications found on univariable analysis included lower height and weight z-scores, and a trend towards increasing age (Table). After controlling for confounders, such as age, BMI z-score, and needle gauge, a diagnosis of NAFLD was associated with a 3-fold decreased odds of complications (OR=0.23, p=0.05)
CHOLESTATIC LIVER INJURY IS EXACERBATED BY PRIOR EXPOSURE TO GULF WAR ILLNESS-RELATED CHEMICALS Matthew McMillin, Stephanie Grant, Gabriel Frampton, Maheedhar Kodali, Ashok Shetty, Sharon DeMorrow Gulf war illness (GWI) is a term used to describe a chronic multisymptom disorder affecting military veterans of the 1990-91 Persian Gulf War. Such symptoms include chronic fatigue, muscle pain, rashes and cognitive problems. GWI has been linked to exposure to a combination of chemicals including the anti-nerve gas drug pyridostigmine bromide (PB) and pesticides (eg DEET or permethrin) with a combination of all of these chemicals likely increasing pathogenesis. While no evidence exists that exposure to GWI-related chemicals causes any particular liver pathology, there is circumstantial evidence to suggest that exposure may reduce the ability of the liver to cope and repair itself after chronic liver injury. The aim of this study is to assess the effects of GWI-related chemicals on cholangiocyte proliferation and liver fibrosis after bile duct ligation (BDL). Methods: Male Sprague Dawley rats were treated with PB (2mg/kg/day; gavage), DEET (60 mg/kg/day, dermal application) and Permethrin (0.2 mg/kg/day) followed by manual restraint stress (15 minutes) daily for 28 consecutive days. Ten weeks after the last treatment, GWI rats or naïve age-matched controls underwent BDL (or sham) surgeries. Cholangiocyte proliferation and intrahepatic bile duct mass (IBDM) was assessed in vivo by immunohistochemistry and qPCR using proliferating cellular nuclear antigen (PCNA) or cytokeratin 19 (CK19) antibodies respectively. Serum chemistry for AST, ALT, alkaline phosphatase and bilirubin were assessed. Proinflammatory cytokine expression was assessed by qPCR and EIA. Liver fibrosis was measured by Sirius red staining and qPCR in total liver extracts for markers of fibrosis and activated hepatic
AASLD Abstracts
S-1064