Single-cell landscape of functionally cured chronic hepatitis B patients reveals activation of innate and altered CD4-CTL-driven adaptive immunity.

BACKGROUND & AIMS: Hepatitis B surface antigen (HBsAg) loss or functional cure (FC) is considered the optimal therapeutic outcome for patients with chronic hepatitis B (CHB). However, the immune-pathological biomarkers and underlying mechanisms of FC remain unclear. In this study we comprehensively interrogate disease-associated cell states identified within intrahepatic tissue and matched PBMCs (peripheral blood mononuclear cells) from patients with CHB or after FC, at the resolution of single cells, to provide novel insights into putative mechanisms underlying FC. METHODS: We combined single-cell transcriptomics (single-cell RNA sequencing) with multiparametric flow cytometry-based immune phenotyping, and multiplexed immunofluorescence to elucidate the immunopathological cell states associated with CHB vs. FC. RESULTS: We found that the intrahepatic environment in CHB and FC displays specific cell identities and molecular signatures that are distinct from those found in matched PBMCs. FC is associated with the emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes, and an activated innate response represented by liver-resident natural killer cells, specific Kupffer cell subtypes and marginated neutrophils. Surprisingly, we found MHC class II-expressing hepatocytes in patients achieving FC, as well as low but persistent levels of covalently closed circular DNA and pregenomic RNA, which may play an important role in FC. CONCLUSIONS: Our study provides conceptually novel insights into the immuno-pathological control of HBV cure, and opens exciting new avenues for clinical management, biomarker discovery and therapeutic development. We believe that the discoveries from this study, as it relates to the activation of an innate and altered immune response that may facilitate sustained, low-grade inflammation, may have broader implications in the resolution of chronic viral hepatitis. IMPACT AND IMPLICATIONS: This study dissects the immuno-pathological cell states associated with functionally cured chronic hepatitis B (defined by the loss of HBV surface antigen or HBsAg). We identified the sustained presence of very low viral load, accessory antigen-presenting hepatocytes, adaptive-memory-like natural killer cells, and the emergence of helper CD4 T cells with cytotoxic or effector-like signatures associated with functional cure, suggesting previously unsuspected alterations in the adaptive immune response, as well as a key role for the innate immune response in achieving or maintaining functional cure. Overall, the insights generated from this study may provide new avenues for the development of alternative therapies as well as patient surveillance for better clinical management of chronic hepatitis B.

Trans-interaction of risk loci 6p24.1 and 10q11.21 is associated with endothelial damage in coronary artery disease.

BACKGROUND AND AIMS: Single nucleotide polymorphism rs6903956 has been identified as one of the genetic risk factors for coronary artery disease (CAD). However, rs6903956 lies in a non-coding locus on chromosome 6p24.1. We aim to interrogate the molecular basis of 6p24.1 containing rs6903956 risk alleles in endothelial disease biology. METHODS AND RESULTS: We generated induced pluripotent stem cells (iPSCs) from CAD patients (AA risk genotype at rs6903956) and non-CAD subjects (GG non-risk genotype at rs6903956). CRISPR-Cas9-based deletions (Δ63-89bp) on 6p24.1, including both rs6903956 and a short tandem repeat variant rs140361069 in linkage disequilibrium, were performed to generate isogenic iPSC-derived endothelial cells. Edited CAD endothelial cells, with removal of 'A' risk alleles, exhibited a global transcriptional downregulation of pathways relating to abnormal vascular physiology and activated endothelial processes. A CXC chemokine ligand on chromosome 10q11.21, CXCL12, was uncovered as a potential effector gene in CAD endothelial cells. Underlying this effect was the preferential inter-chromosomal interaction of 6p24.1 risk locus to a weak promoter of CXCL12, confirmed by chromatin conformation capture assays on our iPSC-derived endothelial cells. Functionally, risk genotypes AA/AG at rs6903956 were associated significantly with elevated levels of circulating damaged endothelial cells in CAD patients. Circulating endothelial cells isolated from patients with risk genotypes AA/AG were also found to have 10 folds higher CXCL12 transcript copies/cell than those with non-risk genotype GG. CONCLUSIONS: Our study reveals the trans-acting impact of 6p24.1 with another CAD locus on 10q11.21 and is associated with intensified endothelial injury.

Human Stem Cell-Derived Endothelial-Hepatic Platform for Efficacy Testing of Vascular-Protective Metabolites from Nutraceuticals.

Atherosclerosis underlies many cardiovascular and cerebrovascular diseases. Nutraceuticals are emerging as a therapeutic moiety for restoring vascular health. Unlike small-molecule drugs, the complexity of ingredients in nutraceuticals often confounds evaluation of their efficacy in preclinical evaluation. It is recognized that the liver is a vital organ in processing complex compounds into bioactive metabolites. In this work, we developed a coculture system of human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and human pluripotent stem cell-derived hepatocytes (hPSC-HEPs) for predicting vascular-protective effects of nutraceuticals. To validate our model, two compounds (quercetin and genistein), known to have anti-inflammatory effects on vasculatures, were selected. We found that both quercetin and genistein were ineffective at suppressing inflammatory activation by interleukin-1ß owing to limited metabolic activity of hPSC-ECs. Conversely, hPSC-HEPs demonstrated metabolic capacity to break down both nutraceuticals into primary and secondary metabolites. When hPSC-HEPs were cocultured with hPSC-ECs to permit paracrine interactions, the continuous turnover of metabolites mitigated interleukin-1ß stimulation on hPSC-ECs. We observed significant reductions in inflammatory gene expressions, nuclear translocation of nuclear factor κB, and interleukin-8 production. Thus, integration of hPSC-HEPs could accurately reproduce systemic effects involved in drug metabolism in vivo to unravel beneficial constituents in nutraceuticals. This physiologically relevant endothelial-hepatic platform would be a great resource in predicting the efficacy of complex nutraceuticals and mechanistic interrogation of vascular-targeting candidate compounds. Stem Cells Translational Medicine 2017;6:851-863.

Computational analysis reveals the coupling between bistability and the sign of a feedback loop in a TGF-ß1 activation model.

BACKGROUND: Bistable behaviors are prevalent in cell signaling and can be modeled by ordinary differential equations (ODEs) with kinetic parameters. A bistable switch has recently been found to regulate the activation of transforming growth factor-ß1 (TGF-ß1) in the context of liver fibrosis, and an ordinary differential equation (ODE) model was published showing that the net activation of TGF-ß1 depends on the balance between two antagonistic sub-pathways. RESULTS: Through modeling the effects of perturbations that affect both sub-pathways, we revealed that bistability is coupled with the signs of feedback loops in the model. We extended the model to include calcium and Krüppel-like factor 2 (KLF2), both regulators of Thrombospondin-1 (TSP1) and Plasmin (PLS). Increased levels of extracellular calcium, which alters the TSP1-PLS balance, would cause high levels of TGF-ß1, resembling a fibrotic state. KLF2, which suppresses production of TSP1 and plasminogen activator inhibitor-1 (PAI1), would eradicate bistability and preclude the fibrotic steady-state. Finally, the loop PLS - TGF-ß1 - PAI1 had previously been reported as negative feedback, but the model suggested a stronger indirect effect of PLS down-regulating PAI1 to produce positive (double-negative) feedback in a fibrotic state. Further simulations showed that activation of KLF2 was able to restore negative feedback in the PLS - TGF-ß1 - PAI1 loop. CONCLUSIONS: Using the TGF-ß1 activation model as a case study, we showed that external factors such as calcium or KLF2 can induce or eradicate bistability, accompanied by a switch in the sign of a feedback loop (PLS - TGF-ß1 - PAI1) in the model. The coupling between bistability and positive/negative feedback suggests an alternative way of characterizing a dynamical system and its biological implications.

Hepatic stellate cell-targeted delivery of hepatocyte growth factor transgene via bile duct infusion enhances its expression at fibrotic foci to regress dimethylnitrosamine-induced liver fibrosis.

Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-ß1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

HGF regulates the activation of TGF-ß1 in rat hepatocytes and hepatic stellate cells.

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor-beta 1 (TGF-ß1). Specifically, we tested whether HGF decreases the levels of active TGF-ß1, and whether such decrease depends on the predominantly hepatocyte-secreted protease plasmin, and whether it depends on the TGF-ß1 activator thrombospondin-1 (TSP-1). With hepatocyte monocultures, we found HGF-induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI-1, TGF-ß1, and TIMP-2). With in vitro models of fibrotic liver (HSC-T6 hepatic stellate cells, or co-cultures of HSC-T6 and hepatocytes), we found high levels of fibrosis-associated proteins such as TSP-1, active TGF-ß1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP-1 protein cleavage; and decreased the levels of active TGF-ß1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF-ß1 activation and Collagen I. Meanwhile, the TSP-1-specific peptide inhibitor, LSKL, reduced TGF-ß1 to the same level as in the HGF-treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP-1 dependent pathway of TGF-ß1 activation. Therefore, HGF can decrease TGF-ß1 activation and TGF-ß1-dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP-1-dependent activation of TGF-ß1.

Plasmin triggers a switch-like decrease in thrombospondin-dependent activation of TGF-ß1.

Transforming growth factor-ß1 (TGF-ß1) is a potent regulator of extracellular matrix production, wound healing, differentiation, and immune response, and is implicated in the progression of fibrotic diseases and cancer. Extracellular activation of TGF-ß1 from its latent form provides spatiotemporal control over TGF-ß1 signaling, but the current understanding of TGF-ß1 activation does not emphasize cross talk between activators. Plasmin (PLS) and thrombospondin-1 (TSP1) have been studied individually as activators of TGF-ß1, and in this work we used a systems-level approach with mathematical modeling and in vitro experiments to study the interplay between PLS and TSP1 in TGF-ß1 activation. Simulations and steady-state analysis predicted a switch-like bistable transition between two levels of active TGF-ß1, with an inverse correlation between PLS and TSP1. In particular, the model predicted that increasing PLS breaks a TSP1-TGF-ß1 positive feedback loop and causes an unexpected net decrease in TGF-ß1 activation. To test these predictions in vitro, we treated rat hepatocytes and hepatic stellate cells with PLS, which caused proteolytic cleavage of TSP1 and decreased activation of TGF-ß1. The TGF-ß1 activation levels showed a cooperative dose response, and a test of hysteresis in the cocultured cells validated that TGF-ß1 activation is bistable. We conclude that switch-like behavior arises from natural competition between two distinct modes of TGF-ß1 activation: a TSP1-mediated mode of high activation and a PLS-mediated mode of low activation. This switch suggests an explanation for the unexpected effects of the plasminogen activation system on TGF-ß1 in fibrotic diseases in vivo, as well as novel prognostic and therapeutic approaches for diseases with TGF-ß dysregulation.

Purpose-driven biomaterials research in liver-tissue engineering.

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.