Adam Arterberry, PhD
Associate Research Scientist, Department of Pediatrics
Project Title: Inflammasome, Unfolded Protein Response, and T cell Immunity in DAIH
Chronic liver allograft dysfunction is a leading cause of patient morbidity and late allograft loss after liver transplantation. An Important cause of chronic liver allograft dysfunction is de novo autoimmune hepatitis (DAIH) seen in 4-7% of pediatric and adult liver transplant recipients. The long term goal of the proposed research is to provide insights into the etiology and pathogenesis of DAIH to inform prevention and management strategies. The main objective of this project is to begin to identify the molecular mechanisms through which monocytes induce Tregs from patients with DAIH to differentiate towards a pro-inflammatory phenotype and lose suppressive function. The rationale is that identification of intrinsic cellular pathways involved in Treg dysfunction in DAIH could lead to potentially new therapies, improve patient care and lower health care costs. If our hypothesis is correct, completing the specific aims will provide attractive new targets for preventive and therapeutic interventions in addition to advancing our understanding of the pathogenesis of DAIH. We will investigate if inflammasome activation (IL-1b, IL-18, caspase-1, and NLRP3) in CD14+ monocytes from patients with DAIH drives the enhanced production of pro-inflammatory polarizing cytokines (IL-12/IL-6). In addition, we will assess whether inflammasome activation in monocytes drives Treg polarization. We seek to determine if the inflammasome induces proinflammatory signaling in monocytes/macrophages from patients with DAIH; and determine if activation of the UPR via ATF6 induces a pro-inflammatory signature in Tregs from patients with DAIH and drives reduced regulatory function. Gene silencing of caspase-1 in CD14+ monocytes and ATF6 in CD127-CD25highCD4+ Tregs using lentiviral shRNA will allow us to (i) confirm activation of the inflammasome (caspase-1, (pro)IL-1b, (pro)IL-18, and NLRP3) in DAIH and establish if inhibition of the inflammasome in monocytes influences the subsequent production of effector cytokines in Tregs when co-cultured and (ii) to verify a functional role for ATF6 in the production of pro-inflammatory cytokines by Tregs, and establish if UPR activation compromises the regulatory capacity of Tregs.
Romina Fiorotto, PhD
Associate Research Scientist, Department of Digestive Diseases
Project Title: CFTR-defective biliary cells from h-iPSCs as a model to study the role of innate in cystic fibrosis liver disease (CFLD)
Cystic fibrosis (CF) is a common and severe genetic disease, caused by mutations in CFTR, a protein that regulates fluid secretion in a number of organs. In the liver, CFTR is expressed in the biliary epithelium, where it promotes the transmembrane efflux of chloride and bicarbonate. A percentage of patients with CF present liver disease (CFLD), a chronic cholangiopathy that can compromise survival and quality of life.
Using an animal model of CF we have demonstrated that lack of CFTR has a profound impact on the innate immunity and on the cytoskeletal architecture of biliary cells and we have identified the protein tyrosine kinase Src as an important target.
A major obstacle to design new therapies is the lack of an experimental model that matches the human CF biliary phenotype. In this project we will exploit the novel technology of human induced pluripotent stem cells (iPSC) to translate our knowledge and the therapeutic applications in a model that closely resemble the human disease.
During the first year of the project we have developed a protocol for differentiation of human iPSCs, derived from a healthy control and from a CF patient with ΔF508CFTR mutation, into mature and functional biliary cells. Preliminary evidence shows that the mutated cholangiocytes present a similar phenotype compare to the mouse model previously described (i.e increased TLR4/NF-kB response, increased Src activation, altered F-actin distribution).
Based on our findings, during the second year of the project we will test in iPSC-derived biliary cell model new therapeutic approaches to target inflammation and restore the integrity of the actin cytoskeleton. The integrity of the actin cytoskeleton is crucial for the stability of CFTR protein at the membrane. In combination with the small molecules that partially correct the ΔF508 defect (i.e VX-809 and VX-770), we might improve CFTR recycling and retention at the plasma membrane therefore increasing the efficacy of the therapies in use to correct the basic ΔF508 defect.
Arya Mani, MD
Associate Professor of Medicine and Genetics, Department of Cardiovascular Medicine
Project Title: The role of Wnt/TCF7L2 in regulation of Liver Fat, Inflammation, and Fibrosis
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, which begins with steatosis and advances to steatohepatitis (NASH), steatofibrosis, and cirrhosis. The pathways involved in disease progression are not well understood. Loss-of-function mutations in Wnt coreceptor LDL receptor-related protein 6 (LRP6) underlie NASH and metabolic syndrome in humans (Mani, Science 20071). Mice with the human disease-associated LRP6R611C mutation exhibit hyperlipidemia, steatohepatitis and steatofibrosis. These traits are associated with increased activation of the noncanonical Wnt pathways and its downstream kinase NLK, which phosphorylates Wnt transcription cofactor TCF7L2 and results in its ubiquitination. The causal link between altered Wnt/TCF7L2 signaling, hyperlipidemia and NASH is supported by normalization of TCF7L2 expression and the rescue of the hyperlipidemia and NASH upon Wnt3a administration to LRP6mut/mut mice. These studies identify diverse disease pathways that underlie a spectrum of NASH-related liver diseases and are triggered by a single human genetic mutation and underscore the importance of TCF7L2 in protection against NASH. Most notable, genetic variants in TCF7L2 gene have been associated with increased risk for NAFLD3,4 and diabetes. Our current focus is on studying the role of TCF7L2 in regulation of liver fat, inflammation and fibrosis and plasma lipids in mice overexpressing TCF7L2 and mice haploinsufficient for TCF7L2.
Dana Peters, PhD
Assistant Professor, Department of Radiology and Biomedical Imaging
Project Title: Magnetic Resonance Biomarkers of Acidosis in the Liver Tumor
Liver cancer is the only cancer with growing incidence rates worldwide, with over 800,000 new cases every year of primary and 300,000 cases of metastatic disease. Transarterial chemoembolization (TACE) is a minimally invasive, image-guided, catheter-based therapy which significantly improves patient survival. However, TACE is still only palliative but could possibly become curative if problems due to imperfect drug delivery—especially for large tumors—were solved. Multiparameter MRI (mpMRI) –MRI which combines several measures of the tumor--could ultimately guide therapies. Our team combines unique expertise in liver cancer therapies, and MR imaging. Liver tumor progression is known to occur in acidic tumor microenvironments (1,2) which stimulate vascularity. Increased cellularity is also a marker of tumor progression. Vascularity, cellularity, and importantly, acidity, can be quantified using MRI methodologies. We are developing mpMRI tools to quantify acidity, vascularity, cellularity, with the goal of employing them to identify post-TACE response of increased tumor aggression, in future studies.
Carol Soroka, PhD
Senior Research Scientist, Department of Digestive Diseases
Project Title: Use of Human Organoids in the Study of Liver Disease: A Model for Maintaining PSC Patient Progenitor Cells in Long-term Culture with the Potential Ability to Screen for the Efficacy of Drug Therapies
Primary sclerosing cholangitis (PSC) is a rare, progressive, and often fatal cholestatic liver disease of unknown etiology. Understanding the mechanisms of the disease and how to treat it is hampered by the diversity seen in the clinical population, the rarity of the disease, and the lack of a reproducible in vitro culture system that can focus on the bile duct cell that represents a very small percent of all hepatic cells. In this Pilot grant study, we will isolate and culture progenitor cells (organoids) directly from the liver of patients with PSC.These cells have a biliary phenotype and can be maintained long-term in culture. Furthermore, they can be frozen down in order to create a biobank of cells that can be used for future studies, including pharmacotherapeutic testing of drugs. The organoids from controls (healthy and diseased) and PSC patients will be compared by genetic analysis and drug therapies will be tested in a personalized fashion, tailored to the phenotype-genotype detected. It is the hope that the use of organoids will provide insight into the diversity seen in the patient population and allow future studies into the mechanisms of this rare disease.