Primary Infection by E. multilocularis Induces Distinct Patterns of Cross Talk between Hepatic Natural Killer T Cells and Regulatory T Cells in Mice.

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans, with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models, indicating that an appropriate adaptive immune response is required for the control of the disease. However, cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients, the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points, downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice.

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus.

Background & Aims: High-dose irradiation is an essential tool to help control the growth of hepatic tumors, but it can cause radiation-induced liver disease (RILD). This life-threatening complication manifests itself months following radiation therapy and is characterized by fibrosis of the pericentral sinusoids. In this study, we aimed to establish a mouse model of RILD to investigate the underlying mechanism of radiation-induced liver fibrosis. Methods: Using a small animal image-guided radiation therapy platform, an irradiation scheme delivering 50 Gy as a single dose to a focal point in mouse livers was designed. Tissues were analyzed 1 and 6 days, and 6 and 20 weeks post-irradiation. Irradiated livers were assessed by histology, immunohistochemistry, imaging mass cytometry and RNA sequencing. Mitochondrial function was assessed using high-resolution respirometry. Results: At 6 and 20 weeks post-irradiation, pericentral fibrosis was visible in highly irradiated areas together with immune cell infiltration and extravasation of red blood cells. RNA sequencing analysis showed gene signatures associated with acute DNA damage, p53 activation, senescence and its associated secretory phenotype and fibrosis. Moreover, gene profiles of mitochondrial damage and an increase in mitochondrial DNA heteroplasmy were detected. Respirometry measurements of hepatocytes in vitro confirmed irradiation-induced mitochondrial dysfunction. Finally, the highly irradiated fibrotic areas showed markers of reactive oxygen species such as decreased glutathione and increased lipid peroxides and a senescence-like phenotype. Conclusions: Based on our mouse model of RILD, we propose that irradiation-induced mitochondrial DNA instability contributes to the development of fibrosis via the generation of excessive reactive oxygen species, p53 pathway activation and a senescence-like phenotype. Lay summary: Irradiation is an efficient cancer therapy, however, its applicability to the liver is limited by life-threatening radiation-induced hepatic fibrosis. We have developed a new mouse model of radiation-induced liver fibrosis, that recapitulates the human disease. Our model highlights the role of mitochondrial DNA instability in the development of irradiation-induced liver fibrosis. This new model and subsequent findings will help increase our understanding of the hepatic reaction to irradiation and to find strategies that protect the liver, enabling the expanded use of radiotherapy to treat hepatic tumors.

Loss of Claudin-3 Impairs Hepatic Metabolism, Biliary Barrier Function, and Cell Proliferation in the Murine Liver.

BACKGROUND & AIMS: Tight junctions in the liver are essential to maintain the blood-biliary barrier, however, the functional contribution of individual tight junction proteins to barrier and metabolic homeostasis remains largely unexplored. Here, we describe the cell type-specific expression of tight junction genes in the murine liver, and explore the regulation and functional importance of the transmembrane protein claudin-3 in liver metabolism, barrier function, and cell proliferation. METHODS: The cell type-specific expression of hepatic tight junction genes is described using our mouse liver single-cell sequencing data set. Differential gene expression in Cldn3-/- and Cldn3+/+ livers was assessed in young and aged mice by RNA sequencing (RNA-seq), and hepatic tissue was analyzed for lipid content and bile acid composition. A surgical model of partial hepatectomy was used to induce liver cell proliferation. RESULTS: Claudin-3 is a highly expressed tight junction protein found in the liver and is expressed predominantly in hepatocytes and cholangiocytes. The histology of Cldn3-/- livers showed no overt phenotype, and the canalicular tight junctions appeared intact. Nevertheless, by RNA-seq we detected a down-regulation of metabolic pathways in the livers of Cldn3-/- young and aged mice, as well as a decrease in lipid content and a weakened biliary barrier for primary bile acids, such as taurocholic acid, taurochenodeoxycholic acid, and taurine-conjugated muricholic acid. Coinciding with defects in the biliary barrier and lower lipid metabolism, there was a diminished hepatocyte proliferative response in Cldn3-/- mice after partial hepatectomy. CONCLUSIONS: Our data show that, in the liver, claudin-3 is necessary to maintain metabolic homeostasis, retention of bile acids, and optimal hepatocyte proliferation during liver regeneration. The RNA-seq data set can be accessed at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159914.

Identification of transcription factors controlling floral morphology in wild Petunia species with contrasting pollination syndromes.

Adaptation to different pollinators is an important driver of speciation in the angiosperms. Genetic approaches such as QTL mapping have been successfully used to identify the underlying speciation genes. However, these methods are often limited by widespread suppression of recombination due to divergence between species. While the mutations that caused the interspecific differences in floral color and scent have been elucidated in a variety of plant genera, the genes that are responsible for morphological differences remain mostly unknown. Differences in floral organ length determine the pollination efficiency of hawkmoths and hummingbirds, and therefore the genes that control these differences are potential speciation genes. Identifying such genes is challenging, especially in non-model species and when studying complex traits for which little prior genetic and biochemical knowledge is available. Here we combine transcriptomics with detailed growth analysis to identify candidate transcription factors underlying interspecific variation in the styles of Petunia flowers. Starting from a set of 2284 genes, stepwise filtering for expression in styles, differential expression between species, correlation with growth-related traits, allele-specific expression in interspecific hybrids, and/or high-impact polymorphisms resulted in a set of 43 candidate speciation genes. Validation by virus-induced gene silencing identified two MYB transcription factors, EOBI and EOBII, that were previously shown to regulate floral scent emission, a trait associated with pollination by hawkmoths.