A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver’s natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450–mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.
Soon Seng Ng, Anming Xiong, Khanh Nguyen, Marilyn Masek, Da Yoon No, Menashe Elazar, Eyal Shteyer, Mark A. Winters, Amy Voedisch, Kate Shaw, Sheikh Tamir Rashid, Curtis W. Frank, Nam Joon Cho, Jeffrey S. Glenn
Intestinal tuft cells are a rare, poorly understood cell type recently shown to be a critical mediator of type 2 immune response to helminth infection. Here, we present advances in segmentation algorithms and analytical tools for multiplex immunofluorescence (MxIF), a platform that enables iterative staining of over 60 antibodies on a single tissue section. These refinements have enabled a comprehensive analysis of tuft cell number, distribution, and protein expression profiles as a function of anatomical location and physiological perturbations. Based solely on DCLK1 immunoreactivity, tuft cell numbers were similar throughout the mouse small intestine and colon. However, multiple subsets of tuft cells were uncovered when protein coexpression signatures were examined, including two new intestinal tuft cell markers, Hopx and EGFR phosphotyrosine 1068. Furthermore, we identified dynamic changes in tuft cell number, composition, and protein expression associated with fasting and refeeding and after introduction of microbiota to germ-free mice. These studies provide a foundational framework for future studies of intestinal tuft cell regulation and demonstrate the utility of our improved MxIF computational methods and workflow for understanding cellular heterogeneity in complex tissues in normal and disease states.
Eliot T. McKinley, Yunxia Sui, Yousef Al-Kofahi, Bryan A. Millis, Matthew J. Tyska, Joseph T. Roland, Alberto Santamaria-Pang, Christina L. Ohland, Christian Jobin, Jeffrey L. Franklin, Ken S. Lau, Michael J. Gerdes, Robert J. Coffey
Unconjugated bilirubin (UCB), a product of heme oxidation, has known immunosuppressant properties but the molecular mechanisms, other than antioxidant effects, remain largely unexplored. We note that UCB modulates T helper type 17 (Th17) immune responses, in a manner dependent upon heightened expression of CD39 ectonucleotidase. UCB has protective effects in experimental colitis, where it enhances recovery after injury and preferentially boosts IL-10 production by colonic intraepithelial CD4+ cells. In vitro, UCB confers immunoregulatory properties on human control Th17 cells, as reflected by increased levels of FOXP3 and CD39 with heightened cellular suppressor ability. Upregulation of CD39 by Th17 cells is dependent upon ligation of the aryl hydrocarbon receptor (AHR) by UCB. Genetic deletion of CD39, as in
Maria Serena Longhi, Marta Vuerich, Alireza Kalbasi, Jessica E. Kenison, Ada Yeste, Eva Csizmadia, Byron Vaughn, Linda Feldbrugge, Shuji Mitshuhashi, Barbara Wegiel, Leo Otterbein, Alan Moss, Francisco J. Quintana, Simon C. Robson
The discovery of metabolite-phenotype associations may highlight candidate biomarkers and metabolic pathways altered in disease states. We sought to identify novel metabolites associated with obesity and one of its major complications, nonalcoholic fatty liver disease (NAFLD), using a liquid chromatography–tandem mass spectrometry method. In 997 individuals in Framingham Heart Study Generation 3 (FHS Gen 3), we identified an association between anandamide (AEA) and BMI. Further examination revealed that AEA was associated with radiographic hepatic steatosis. In a histologically defined NAFLD cohort, AEA was associated with NAFLD severity, the presence of nonalcoholic steatohepatitis, and fibrosis. These data highlight AEA as a marker linking cardiometabolic disease and NAFLD severity.
W. Taylor Kimberly, John F. O’Sullivan, Anjali K. Nath, Michelle Keyes, Xu Shi, Martin G. Larson, Qiong Yang, Michelle T. Long, Ramachandran Vasan, Randall T. Peterson, Thomas J. Wang, Kathleen E. Corey, Robert E. Gerszten
G protein–coupled receptor 15 (GPR15) was recently highlighted as a colon-homing receptor for murine and human CD4+ T cells. The aim of this study was to explore the functional phenotype of human GPR15+CD4+ T cells, focusing on Tregs and effector T cells (Teffs), and to determine whether GPR15 is the driver for the migration of T cells to the colon during ulcerative colitis (UC). In the peripheral blood, GPR15 was expressed on Tregs and Teffs; both GPR15+ T cell subsets produced less IFN-γ and IL-4 but more IL-17 after stimulation and showed a higher migration activity compared with GPR15–CD4+ T cells. In UC patients, GPR15 expression was increased on Tregs in the peripheral blood but not on Teffs. Interestingly, the expression of GPR15 was significantly enhanced on colonic T cells of UC patients in noninflamed biopsies but not in inflamed biopsies. The differential expression of GPR15 in UC patients was accompanied by a significant reduction of bacterial immunoregulatory metabolites in the feces. In conclusion, GPR15 expression on CD4+ T cells is altered in UC patients, which may have implications for the development of therapeutic approaches to target T cell trafficking to the colon.
Alexandra Adamczyk, Daniel Gageik, Annika Frede, Eva Pastille, Wiebke Hansen, Andreas Rueffer, Jan Buer, Jürgen Büning, Jost Langhorst, Astrid M. Westendorf
Ta-Chiang Liu, Takeo Naito, Zhenqiu Liu, Kelli L. VanDussen, Talin Haritunians, Dalin Li, Katsuya Endo, Yosuke Kawai, Masao Nagasaki, Yoshitaka Kinouchi, Dermot P.B. McGovern, Tooru Shimosegawa, Yoichi Kakuta, Thaddeus S. Stappenbeck
Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor–like receptor (
Reema B. Davis, Daniel O. Kechele, Elizabeth S. Blakeney, John B. Pawlak, Kathleen M. Caron
Biliary atresia is an obstructive cholangiopathy of infancy that progresses to end-stage cirrhosis. Although the pathogenesis of the disease is not completely understood, previous reports link TNFα to apoptosis of the bile duct epithelium in the presence of IFNγ. Here, we investigate if TNFα signaling regulates pathogenic mechanisms of biliary atresia. First, we quantified the expression of
Pranavkumar Shivakumar, Tatsuki Mizuochi, Reena Mourya, Sridevi Gutta, Li Yang, Zhenhua Luo, Jorge A. Bezerra
The fibrotic reaction, which can account for over 70%–80% of the tumor mass, is a characteristic feature of human pancreatic ductal adenocarcinoma (PDAC) tumors. It is associated with activation and proliferation of pancreatic stellate cells (PSCs), which are key regulators of collagen I production and fibrosis in vivo. In this report, we show that members of the bromodomain and extraterminal (BET) family of proteins are expressed in primary PSCs isolated from human PDAC tumors, with BRD4 positively regulating, and BRD2 and BRD3 negatively regulating, collagen I expression in primary cancer-associated PSCs. We show that the inhibitory effect of pan-BET inhibitors on collagen I expression in primary cancer-associated PSCs is through blocking of BRD4 function. Importantly, we show that FOSL1 is repressed by BRD4 in primary cancer-associated PSCs and negatively regulates collagen I expression. While BET inhibitors do not affect viability or induce PSC apoptosis or senescence, BET inhibitors induce primary cancer-associated PSCs to become quiescent. Finally, we show that BET inhibitors attenuate stellate cell activation, fibrosis, and collagen I production in the EL-KrasG12D transgenic mouse model of pancreatic tumorigenesis. Our results demonstrate that BET inhibitors regulate fibrosis by modulating the activation and function of cancer-associated PSCs.
Krishan Kumar, Brian T. DeCant, Paul J. Grippo, Rosa F. Hwang, David J. Bentrem, Kazumi Ebine, Hidayatullah G. Munshi
GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn’s disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis.
Joanna M. Peloquin, Gautam Goel, Lingjia Kong, Hailiang Huang, Talin Haritunians, R. Balfour Sartor, Mark J. Daly, Rodney D. Newberry, Dermot P. McGovern, Vijay Yajnik, Sergio A. Lira, Ramnik J. Xavier
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