Our long-term OBJECTIVES are to apply the fundamental concepts and technologies of cell and molecular biology to understand the role of cholangiocytes, the epithelial cells lining intrahepatic bile ducts, in health and disease. Here we concentrate on cholangiocyte recognition and response to pathogens or pathogen-derived products. The biliary tract is periodically exposed to microbial-derived toxins (e.g. lipopolysaccharide) or colonized by pathogenic microorganisms (e.g. Cryptosporidium parvum, a parasitic protozoon and Category B Biodefense pathogen that causes intestinal and biliary cryptosporidiosis). Recent evidence from our lab indicates that: (i) C. parvum infection or treatment of cultured cholangiocytes with the TLR4 agonist, LPS, promotes the rapid activation of the small GTPase, N-Ras;(ii) activated N-Ras contributes to cholangiocyte production of proinflammatory cytokines and pathogen-induced proliferation;(iii) C. parvum and LPS alter the expression of the cholangiocyte microRNA expression profile, including the let-7 family;(iv) manipulation of selected microRNA expression (i.e. let-7i) influences post-transcriptional regulation of targeted proinflammatory mRNAs in cholangiocytes, including N-Ras;and, (v) our in vivo models of biliary responses to microbial stimuli reflect our in vitro observations of increased proinflammatory responses. Thus, we will test the CENTRAL HYPOTHESIS that pathogen-induced TLR4-dependent Ras activation promotes the cholangiocyte innate immune phenotype (cytokine production, increased proliferation) through transcriptional suppression of the let- 7 microRNAs. In our three integrated SPECIFIC AIMS we will test the hypotheses that: (i) cholangiocyte pathogen recognition activates N-Ras through a TLR-dependent mechanism, and activated N-Ras initiates proinflammatory cytokine production;(ii) pathogen-induced N-Ras activation in cholangiocytes suppresses let- 7 microRNA transcription, which promotes an increase in let-7 proinflammatory target proteins;and, (iii) pathogen-induced N-Ras activation and reduction of let-7 promote cholangiocyte proinflammatory responses in vivo and are essential for inflammatory cell infiltration. This proposal incorporates new in vitro techniques and animal models, novel hypotheses regarding the role of proinflammatory signaling axes that regulate and are regulated by microRNAs, and gene regulation by microRNAs in cholangiocyte proinflammatory cytokine expression and proliferation. Our results will yield new mechanistic insights into the normal cholangiocyte functions of recognition and response to microbial pathogens, and may provide a rational basis for the design and implementation of new therapeutic strategies for inflammatory conditions and microbial infection of the biliary tract.

Public Health Relevance

Cholangiocytes are epithelial cells that line intrahepatic bile ducts. These cells are periodically exposed to pathogen-derived toxins (e.g. lipopolysaccharide) or colonized by pathogenic microorganisms (e.g. Cryptosporidium). This proposal focuses on the molecular mechanisms and functional consequences of microbe-induced signaling cascades culminating in a proinflammatory response by cholangiocytes. Our focus will be on microbe recognition-dependent activation of the signaling axis involving the small GTPase, Ras, as well as implications of this activation on the regulated expression of microRNAs, small RNA molecules that regulate gene expression. We propose that this signaling pathway promotes cholangiocyte inflammatory responses, which promotes the attraction of leukocytes to the portal tract.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI089713-01A1
Application #
8099343
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Palker, Thomas J
Project Start
2011-02-15
Project End
2016-01-31
Budget Start
2011-02-15
Budget End
2012-01-31
Support Year
1
Fiscal Year
2011
Total Cost
$354,825
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
O'Hara, Steven P; Splinter, Patrick L; Trussoni, Christy E et al. (2017) ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence-associated Protein Cyclin-dependent Kinase Inhibitor 2A. J Biol Chem 292:4833-4846
Tabibian, James H; O'Hara, Steven P; Trussoni, Christy E et al. (2016) Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis. Hepatology 63:185-96
Tabibian, James H; Varghese, Cyril; LaRusso, Nicholas F et al. (2016) The enteric microbiome in hepatobiliary health and disease. Liver Int 36:480-7
Gradilone, Sergio A; O'Hara, Steven P; Masyuk, Tetyana V et al. (2015) MicroRNAs and benign biliary tract diseases. Semin Liver Dis 35:26-35
Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L et al. (2015) Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR). PLoS One 10:e0125793
O'Hara, Steven P; Gradilone, Sergio A; Masyuk, Tetyana V et al. (2014) MicroRNAs in Cholangiopathies. Curr Pathobiol Rep 2:133-142
Tabibian, James H; O'Hara, Steven P; Lindor, Keith D (2014) Primary sclerosing cholangitis and the microbiota: current knowledge and perspectives on etiopathogenesis and emerging therapies. Scand J Gastroenterol 49:901-8
Tabibian, James H; O'Hara, Steven P; Splinter, Patrick L et al. (2014) Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis. Hepatology 59:2263-75
Tabibian, James H; Trussoni, Christy E; O'Hara, Steven P et al. (2014) Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis. Lab Invest 94:1126-33
Tabibian, James H; Masyuk, Anatoliy I; Masyuk, Tetyana V et al. (2013) Physiology of cholangiocytes. Compr Physiol 3:541-65

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