Biology subjects

Chung, R. T.

Publications and source records attributed to Chung, R. T..

5 recordsLinked to original sources

bioRxiv2023

Precision-Cut Liver Slices as an ex vivo model to evaluate antifibrotic therapies for liver fibrosis and cirrhosis

BackgroundPrecision-Cut Liver Slices (PCLS) are an ex vivo culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximates in vivo pathology than in vitro methods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS. MethodsPCLS of 250 m thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl4). PCLS were treated with 5 M Erlotinib for 72 hours. Histology and gene expression were then compared with in vivo murine experiments and TGF-{beta}1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver. ResultsPCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genes Il6, Col1a1 and Timp1 in PCLS from CDAHFD-induced cirrhotic mice, and Il6, Col1a1 and Tgfb1 in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression of Col1a1, Timp1, Tgfb1 and Il6, which was consistent with the impact of erlotinib on Col1a1 and Tgfb1 expression in in vivo DEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl4-induced cirrhosis caused reduced expression of Timp1, Col1a1 and Tgfb1, which was consistent with the effect of erlotinib in in vivo CCl4-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-{beta}1 treatment upregulated Acta2 (SMA), while treatment with erlotinib inhibited the expression of Acta2. Similar expression results were observed in TGF-{beta}1 treated in vitro HSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples. ConclusionThe responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed in vivo and in vitro. Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).

cell biology↗

bioRxiv2023

Liver-specific Inflammatory Signatures Predict Clinically Significant Liver Damage.

Background and AimsInflammation drives progression of chronic liver disease. However, the triggers of inflammation remain undefined during chronic hepatitis B (CHB) because hepatic flares are spontaneous and difficult to capture. We used nucleoside analogue (NA) withdrawal to investigate early inflammatory events because liver damage after stopping therapy occurs in a predictable time frame. 11 CHB patients underwent 192 weeks of NA therapy before a protocol defined stop. Liver fine-needle aspirates (FNAs) were collected at baseline and 4-weeks post-withdrawal and analyzed using flow cytometry and single-cell RNA sequencing (scRNA-seq). Intrahepatic mononuclear cells (IHMCs) from uninfected livers were used to validate transcriptomic findings. At 4 weeks post NA-withdrawal, HBV DNA rebounded but alanine aminotransferase (ALT) levels remained normal, 7/11 patients developed ALT elevations (>2xULN) at later timepoints. There were no changes in cell frequencies between baseline and viral rebound. ScRNA-seq revealed upregulation of IFN stimulated genes (ISGs) and pro-inflammatory cytokine MIF upon viral rebound. In vitro experiments confirmed the type I IFN-dependent ISG profile whereas MIF was induced primarily by IL-12. MIF exposure further amplified inflammatory cytokine production by myeloid cells. Our data show that innate immune activation is detectable in the liver before clinically-significant liver damage is detectable in the serum.

immunology↗

bioRxiv2021

Clinical implementation of single-cell RNA sequencing using liver fine needle aspirate tissue sampling and centralized processing captures compartment specific immuno-diversity

Blood samples are frequently collected in human studies of the immune system but poorly represent tissue-resident immunity. Understanding the immunopathogenesis of tissue-restricted diseases, such as chronic hepatitis B, necessitates direct investigation of local immune responses. We developed a workflow that enables frequent, minimally invasive collection of liver fine-needle aspirates in multi-site international studies and centralized single-cell RNA sequencing data generation using the Seq-Well S3 picowell-based technology. All immunological cell types were captured, including liver macrophages, and showed distinct compartmentalization and transcriptional profiles, providing a systematic assessment of the capabilities and limitations of peripheral blood samples when investigating tissue-restricted diseases. The ability to electively sample the liver of chronic viral hepatitis patients and generate high-resolution data will enable multi-site clinical studies to power fundamental and therapeutic discovery.

immunology↗

bioRxiv2021

Differentiation of exhausted CD8 T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Analysis of antigen-specific CD8+ T cells before and after antigen removal in human hepatitis C virus (HCV) infection confirmed pervasive phenotypic, functional, and transcriptional differences between exhausted and memory CD8+ T cells. After viral cure, we observed broad phenotypic and transcriptional changes in clonally stable exhausted T-cell populations suggesting differentiation towards a memory-like profile. However, functionally, the cells showed little improvement and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for shorter periods of time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved acute HCV infection. Thus, duration of T cell stimulation impacts the ability to recover from exhaustion, as antigen removal after long-term T cell exhaustion is insufficient for the development of key T cell memory characteristics.

immunology↗

bioRxiv2021

Bile salt hydrolases deplete conjugated bile acids and erode gut barrier integrity in non-alcoholic steatohepatitis

Altered host-microbe interactions and increased intestinal permeability have been implicated in the pathogenesis of a range of diseases. However, the mechanisms by which gut microbes affect epithelial barrier integrity remain unclear. Few host-produced metabolites that protect against epithelial damage have been identified, and whether microbial metabolism of host factors alters intestinal barrier function is unknown. Here, we investigate the effects of bacterial metabolism of host-produced bile acid (BA) metabolites on epithelial barrier integrity. We observe that rats fed a choline-deficient, high-fat diet (CDAHFD) exhibit reduced abundance of host-produced conjugated BAs in the intestine at early timepoints coinciding with increased permeability. We show that in vitro, conjugated BAs protect gut epithelial monolayers from damage caused by bacterially produced unconjugated BAs through micelle formation. We then demonstrate that inhibition of BA deconjugation using a small molecule inhibitor of gut bacterial bile salt hydrolase (BSH) enzymes prevents development of pathologic intestinal permeability and hepatic inflammation in CDAHFD-fed rats. Finally, we show that the predominant conjugated BAs in humans protect against epithelial barrier disruption in vitro. Our study identifies a protective role for conjugated BAs in intestinal epithelial barrier function and suggests that rational manipulation of microbial BA metabolism could be leveraged to regulate gut barrier integrity.

physiology↗