Priming and Maintenance of Adaptive Immunity in the Liver.

The liver's unique characteristics have a profound impact on the priming and maintenance of adaptive immunity. This review delves into the cellular circuits that regulate adaptive immune responses in the liver, with a specific focus on hepatitis B virus infection as an illustrative example. A key aspect highlighted is the liver's specialized role in priming CD8+ T cells, leading to a distinct state of immune hyporesponsiveness. Additionally, the influence of the liver's hemodynamics and anatomical features, particularly during liver fibrosis and cirrhosis, on the differentiation and function of adaptive immune cells is discussed. While the primary emphasis is on CD8+ T cells, recent findings regarding the involvement of B cells and CD4+ T cells in hepatic immunity are also reviewed. Furthermore, we address the challenges ahead and propose integrating cutting-edge techniques, such as spatial biology, and combining mouse models with human sample analyses to gain comprehensive insights into the liver's adaptive immunity. This understanding could pave the way for novel therapeutic strategies targeting infectious diseases, malignancies, and inflammatory liver conditions like metabolic dysfunction-associated steatohepatitis and autoimmune hepatitis.

Cruel to Be Kind: Epithelial, Microbial, and Immune Cell Interactions in Gastrointestinal Cancers.

A plethora of experimental and epidemiological evidence supports a critical role for inflammation and adaptive immunity in the onset of cancer and in shaping its response to therapy. These data are particularly robust for gastrointestinal (GI) cancers, such as those affecting the GI tract, liver, and pancreas, on which this review is focused. We propose a unifying hypothesis according to which intestinal barrier disruption is the origin of tumor-promoting inflammation that acts in conjunction with tissue-specific cancer-initiating mutations. The gut microbiota and its products impact tissue-resident and recruited myeloid cells that promote tumorigenesis through secretion of growth- and survival-promoting cytokines that act on epithelial cells, as well as fibrogenic and immunosuppressive cytokines that interfere with the proper function of adaptive antitumor immunity. Understanding these relationships should improve our ability to prevent cancer development and stimulate the immune system to eliminate existing malignancies.

New Molecular Insights into Immune Cell Development.

During development innate lymphoid cells and specialized lymphocyte subsets colonize peripheral tissues, where they contribute to organogenesis and later constitute the first line of protection while maintaining tissue homeostasis. A few of these subsets are produced only during embryonic development and remain in the tissues throughout life. They are generated through a unique developmental program initiated in lympho-myeloid-primed progenitors, which lose myeloid and B cell potential. They either differentiate into innate lymphoid cells or migrate to the thymus to give rise to embryonic T cell receptor-invariant T cells. At later developmental stages, adaptive T lymphocytes are derived from lympho-myeloid progenitors that colonize the thymus, while lymphoid progenitors become specialized in the production of B cells. This sequence of events highlights the requirement for stratification in the establishment of immune functions that determine efficient seeding of peripheral tissues by a limited number of cells.

Immune Responses in the Liver.

The liver is a key, frontline immune tissue. Ideally positioned to detect pathogens entering the body via the gut, the liver appears designed to detect, capture, and clear bacteria, viruses, and macromolecules. Containing the largest collection of phagocytic cells in the body, this organ is an important barrier between us and the outside world. Importantly, as portal blood also transports a large number of foreign but harmless molecules (e.g., food antigens), the liver's default immune status is anti-inflammatory or immunotolerant; however, under appropriate conditions, the liver is able to mount a rapid and robust immune response. This balance between immunity and tolerance is essential to liver function. Excessive inflammation in the absence of infection leads to sterile liver injury, tissue damage, and remodeling; insufficient immunity allows for chronic infection and cancer. Dynamic interactions between the numerous populations of immune cells in the liver are key to maintaining this balance and overall tissue health.

First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Identifying specific functional roles for senescence across cell types.

Cellular senescence plays critical roles in aging, regeneration, and disease; yet, the ability to discern its contributions across various cell types to these biological processes remains limited. In this study, we generated an in vivo genetic toolbox consisting of three p16Ink4a-related intersectional genetic systems, enabling pulse-chase tracing (Sn-pTracer), Cre-based tracing and ablation (Sn-cTracer), and gene manipulation combined with tracing (Sn-gTracer) of defined p16Ink4a+ cell types. Using liver injury and repair as an example, we found that macrophages and endothelial cells (ECs) represent distinct senescent cell populations with different fates and functions during liver fibrosis and repair. Notably, clearance of p16Ink4a+ macrophages significantly mitigates hepatocellular damage, whereas eliminating p16Ink4a+ ECs aggravates liver injury. Additionally, targeted reprogramming of p16Ink4a+ ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the functional diversity of p16Ink4a+ cells and offers insights for developing cell-type-specific senolytic therapies in the future.

Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

In vivo DNA replication dynamics unveil aging-dependent replication stress.

The genome duplication program is affected by multiple factors in vivo, including developmental cues, genotoxic stress, and aging. Here, we monitored DNA replication initiation dynamics in regenerating livers of young and old mice after partial hepatectomy to investigate the impact of aging. In young mice, the origin firing sites were well defined; the majority were located 10-50 kb upstream or downstream of expressed genes, and their position on the genome was conserved in human cells. Old mice displayed the same replication initiation sites, but origin firing was inefficient and accompanied by a replication stress response. Inhibitors of the ATR checkpoint kinase fully restored origin firing efficiency in the old mice but at the expense of an inflammatory response and without significantly enhancing the fraction of hepatocytes entering the cell cycle. These findings unveil aging-dependent replication stress and a crucial role of ATR in mitigating the stress-associated inflammation, a hallmark of aging.

Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Structure-guided discovery of bile acid derivatives for treating liver diseases without causing itch.

Chronic itch is a debilitating symptom profoundly impacting the quality of life in patients with liver diseases like cholestasis. Activation of the human G-protein coupled receptor, MRGPRX4 (hX4), by bile acids (BAs) is implicated in promoting cholestasis itch. However, the detailed underlying mechanisms remain elusive. Here, we identified 3-sulfated BAs that are elevated in cholestatic patients with itch symptoms. We solved the cryo-EM structure of hX4-Gq in a complex with 3-phosphated deoxycholic acid (DCA-3P), a mimic of the endogenous 3-sulfated deoxycholic acid (DCA-3S). This structure revealed an unprecedented ligand-binding pocket in MRGPR family proteins, highlighting the crucial role of the 3-hydroxyl (3-OH) group on BAs in activating hX4. Guided by this structural information, we designed and developed compound 7 (C7), a BA derivative lacking the 3-OH. Notably, C7 effectively alleviates hepatic injury and fibrosis in liver disease models while significantly mitigating the itch side effects.

Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease.

Somatic mutations in nonmalignant tissues accumulate with age and injury, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate genes in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to nonalcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side by side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Tbx3, Bcl6, or Smyd2 resulted in protection against hepatic steatosis. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease.

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

Mechanisms and disease consequences of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide. Its more advanced subtype, nonalcoholic steatohepatitis (NASH), connotes progressive liver injury that can lead to cirrhosis and hepatocellular carcinoma. Here we provide an in-depth discussion of the underlying pathogenetic mechanisms that lead to progressive liver injury, including the metabolic origins of NAFLD, the effect of NAFLD on hepatic glucose and lipid metabolism, bile acid toxicity, macrophage dysfunction, and hepatic stellate cell activation, and consider the role of genetic, epigenetic, and environmental factors that promote fibrosis progression and risk of hepatocellular carcinoma in NASH.

Microscopic examination of spatial transcriptome using Seq-Scope.

Spatial barcoding technologies have the potential to reveal histological details of transcriptomic profiles; however, they are currently limited by their low resolution. Here, we report Seq-Scope, a spatial barcoding technology with a resolution comparable to an optical microscope. Seq-Scope is based on a solid-phase amplification of randomly barcoded single-molecule oligonucleotides using an Illumina sequencing platform. The resulting clusters annotated with spatial coordinates are processed to expose RNA-capture moiety. These RNA-capturing barcoded clusters define the pixels of Seq-Scope that are ∼0.5-0.8 µm apart from each other. From tissue sections, Seq-Scope visualizes spatial transcriptome heterogeneity at multiple histological scales, including tissue zonation according to the portal-central (liver), crypt-surface (colon) and inflammation-fibrosis (injured liver) axes, cellular components including single-cell types and subtypes, and subcellular architectures of nucleus and cytoplasm. Seq-Scope is quick, straightforward, precise, and easy-to-implement and makes spatial single-cell analysis accessible to a wide group of biomedical researchers.

Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Glycogen accumulation and phase separation drives liver tumor initiation.

Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

LECT2, a Ligand for Tie1, Plays a Crucial Role in Liver Fibrogenesis.

Liver fibrosis is a very common condition seen in millions of patients with various liver diseases, and yet no effective treatments are available owing to poorly characterized molecular pathogenesis. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2) is a functional ligand of Tie1, a poorly characterized endothelial cell (EC)-specific orphan receptor. Upon binding to Tie1, LECT2 interrupts Tie1/Tie2 heterodimerization, facilitates Tie2/Tie2 homodimerization, activates PPAR signaling, and inhibits the migration and tube formations of EC. In vivo studies showed that LECT2 overexpression inhibits portal angiogenesis, promotes sinusoid capillarization, and worsens fibrosis, whereas these changes were reversed in Lect2-KO mice. Adeno-associated viral vector serotype 9 (AAV9)-LECT2 small hairpin RNA (shRNA) treatment significantly attenuates fibrosis. Upregulation of LECT2 is associated with advanced human liver fibrosis staging. We concluded that targeting LECT2/Tie1 signaling may represent a potential therapeutic target for liver fibrosis, and serum LECT2 level may be a potential biomarker for the screening and diagnosis of liver fibrosis.