Cross-talk between ILC2 and Gata3high Tregs locally constrains adaptive type 2 immunity.

Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.

Polyploidisation pleiotropically buffers ageing in hepatocytes.

BACKGROUND & AIMS: Polyploidy in hepatocytes has been proposed as a genetic mechanism to buffer against transcriptional dysregulation. Here, we aim to demonstrate the role of polyploidy in modulating gene regulatory networks in hepatocytes during ageing. METHODS: We performed single-nucleus RNA sequencing in hepatocyte nuclei of different ploidy levels isolated from young and old wild-type mice. Changes in the gene expression and regulatory network were compared to three independent strains that were haploinsufficient for HNF4A, CEBPA or CTCF, representing non-deleterious perturbations. Phenotypic characteristics of the liver section were additionally evaluated histologically, whereas the genomic allele composition of hepatocytes was analysed by BaseScope. RESULTS: We observed that ageing in wild-type mice results in nuclei polyploidy and a marked increase in steatosis. Haploinsufficiency of liver-specific master regulators (HFN4A or CEBPA) results in the enrichment of hepatocytes with tetraploid nuclei at a young age, affecting the genomic regulatory network, and dramatically suppressing ageing-related steatosis tissue wide. Notably, these phenotypes are not the result of subtle disruption to liver-specific transcriptional networks, since haploinsufficiency in the CTCF insulator protein resulted in the same phenotype. Further quantification of genotypes of tetraploid hepatocytes in young and old HFN4A-haploinsufficient mice revealed that during ageing, tetraploid hepatocytes lead to the selection of wild-type alleles, restoring non-deleterious genetic perturbations. CONCLUSIONS: Our results suggest a model whereby polyploidisation leads to fundamentally different cell states. Polyploid conversion enables pleiotropic buffering against age-related decline via non-random allelic segregation to restore a wild-type genome. IMPACT AND IMPLICATIONS: The functional role of hepatocyte polyploidisation during ageing is poorly understood. Using single-nucleus RNA sequencing and BaseScope approaches, we have studied ploidy dynamics during ageing in murine livers with non-deleterious genetic perturbations. We have identified that hepatocytes present different cellular states and the ability to buffer ageing-associated dysfunctions. Tetraploid nuclei exhibit robust transcriptional networks and are better adapted to genomically overcome perturbations. Novel therapeutic interventions aimed at attenuating age-related changes in tissue function could be exploited by manipulation of ploidy dynamics during chronic liver conditions.

The senescent secretome drives PLVAP expression in cultured human hepatic endothelial cells to promote monocyte transmigration.

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease.

Effect of MMP Inhibitors on the Bond Strength of Fibre Posts After Ageing.

AIM: This in vitro study aimed to evaluate the effect of matrix metalloproteinase (MMP) inhibitors on the bond strength of resin-cemented fibre posts to radicular dentin under an aged-loaded condition. MATERIALS AND METHODS: Radicular dentin was prepared and irrigated by MMP inhibitor solution after root canal obturation in 60 extracted single-rooted teeth based on 6 groups: (1) 2% chlorhexidine (CHX) + loaded; (2) CHX + unloaded; (3) 0.5% benzalkonium chloride (BAC) + loaded; (4) BAC + unloaded; (5) 17% ethylenediaminetetraacetic acid (EDTA) + loaded; and (6) EDTA + unloaded. After final rinsing, all specimens were sliced cross-sectionally and kept in a water bath for 12 months of ageing. Groups 1, 3, and 5 were subjected to cyclic loading. Push-out tests were conducted using a universal testing machine, and failure mode was examined. The data were analysed using 3-way analysis of variance and post hoc tests at α = 0.05. RESULTS: BAC + unloaded demonstrated the highest mean bond strength (3.12 ± 0.18 MPa; P < .001), while the BAC + loaded and CHX + loaded groups showed a significantly lower push-out bond strength than their unloaded counterparts. Mixed adhesive-cohesive failure was the most common failure mode observed. CONCLUSIONS: Without cycling loading, BAC was superior to CHX and EDTA in preserving the bond strength of resin-cemented fibre posts after 12 months of ageing. Loading significantly weakened the effectiveness of BAC and CHX in preserving the bond strength.

Spatial transcriptomics using multiplexed deterministic barcoding in tissue.

Spatially resolved transcriptomics of tissue sections enables advances in fundamental and applied biomedical research. Here, we present Multiplexed Deterministic Barcoding in Tissue (xDBiT) to acquire spatially resolved transcriptomes of nine tissue sections in parallel. New microfluidic chips were developed to spatially encode mRNAs over a total tissue area of 1.17 cm2 with a 50 µm resolution. Optimization of the biochemical protocol increased read and gene counts per spot by one order of magnitude compared to previous reports. Furthermore, the introduction of alignment markers allowed seamless registration of images and spatial transcriptomic spots. Together with technological advances, we provide an open-source computational pipeline to prepare raw sequencing data for downstream analysis. The functionality of xDBiT was demonstrated by acquiring 16 spatially resolved transcriptomic datasets from five different murine organs, including the cerebellum, liver, kidney, spleen, and heart. Factor analysis and deconvolution of spatial transcriptomes allowed for in-depth characterization of the murine kidney.

Isolation of Nuclei from Flash-frozen Liver Tissue for Single-cell Multiomics.

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease. This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

Hepatocyte-specific activity of TSC22D4 triggers progressive NAFLD by impairing mitochondrial function.

OBJECTIVE: Fibrotic organ responses have recently been identified as long-term complications in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date. METHODS: Based on our previous discovery of transforming growth factor beta-like stimulated clone (TSC)22D4 as a key driver of insulin resistance and glucose intolerance in obesity and type 2 diabetes, we generated a TSC22D4-hepatocyte specific knockout line (TSC22D4-HepaKO) and exposed mice to control or NASH diet models. Mechanistic insights were generated by metabolic phenotyping and single-nuclei RNA sequencing. RESULTS: Hepatic TSC22D4 expression was significantly correlated with markers of liver disease progression and fibrosis in both murine and human livers. Indeed, hepatic TSC22D4 levels were elevated in human NASH patients as well as in several murine NASH models. Specific genetic deletion of TSC22D4 in hepatocytes led to reduced liver lipid accumulation, improvements in steatosis and inflammation scores and decreased apoptosis in mice fed a lipogenic MCD diet. Single-nuclei RNA sequencing revealed a distinct TSC22D4-dependent gene signature identifying an upregulation of mitochondrial-related processes in hepatocytes upon loss of TSC22D4. An enrichment of genes involved in the TCA cycle, mitochondrial organization, and triglyceride metabolism underscored the hepatocyte-protective phenotype and overall decreased liver damage as seen in mouse models of hepatocyte-selective TSC22D4 loss-of-function. CONCLUSIONS: Together, our data uncover a new connection between targeted depletion of TSC22D4 and intrinsic metabolic processes in progressive liver disease. Hepatocyte-specific reduction of TSC22D4 improves hepatic steatosis and promotes hepatocyte survival via mitochondrial-related mechanisms thus paving the way for targeted therapies.

COX2 regulates senescence secretome composition and senescence surveillance through PGE2.

Senescent cells trigger their own immune-mediated destruction, termed senescence surveillance. This is dependent on the inflammatory senescence-associated secretory phenotype (SASP), which includes COX2, an enzyme with complex roles in cancer. The role COX2 plays during senescence surveillance is unknown. Here, we show that during RAS-induced senescence (RIS), COX2 is a critical regulator of SASP composition and senescence surveillance in vivo. COX2 regulates the expression of multiple inflammatory SASP components through an autocrine feedback loop involving its downstream product, prostaglandin E2 (PGE2), binding to EP4. During in vivo hepatocyte RIS, Cox2 is critical to tumor suppression, Cxcl1 expression, and immune-mediated senescence surveillance, partially through PGE2. Loss of Cox2 in RIS dysregulates the intrahepatic immune microenvironment, with enrichment of immunosuppressive immature myeloid cells and CD4+ regulatory T lymphocytes. Therefore, COX2 and PGE2 play a critical role in senescence, shaping SASP composition, promoting senescence surveillance and tumor suppression in the earliest stages of tumorigenesis.

NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR.

Exposure of melanocytes to ultraviolet radiation (UVR) induces the formation of UV lesions that can produce deleterious effects in genomic DNA. Encounters of replication forks with unrepaired UV lesions can lead to several complex phenomena, such as the formation of DNA double-strand breaks (DSBs). The NR4A family of nuclear receptors are transcription factors that have been associated with mediating DNA repair functions downstream of the MC1R signaling pathway in melanocytes. In particular, emerging evidence shows that upon DNA damage, the NR4A2 receptor can translocate to sites of UV lesion by mechanisms requiring post-translational modifications within the N-terminal domain and at a serine residue in the DNA-binding domain at position 337. Following this, NR4A2 aids in DNA repair by facilitating chromatin relaxation, allowing accessibility for DNA repair machinery. Using A2058 and HT144 melanoma cells engineered to stably express wild-type or mutant forms of the NR4A2 proteins, we reveal that the expression of functional NR4A2 is associated with elevated cytoprotection against UVR. Conversely, knockdown of NR4A2 expression by siRNA results in a significant loss of cell viability after UV insult. By analyzing the kinetics of the ensuing 53BP1 and RAD51 foci following UV irradiation, we also reveal that the expression of mutant NR4A2 isoforms, lacking the ability to translocate, transactivate, or undergo phosphorylation, display compromised repair capacity.Implications: These data expand the understanding of the mechanism by which the NR4A2 nuclear receptor can facilitate DNA DSB repair. Mol Cancer Res; 15(9); 1184-96. ©2017 AACR.

Nuclear receptor function in skin health and disease: therapeutic opportunities in the orphan and adopted receptor classes.

The skin forms a vital barrier between an organism's external environment, providing protection from pathogens and numerous physical and chemical threats. Moreover, the intact barrier is essential to prevent water and electrolyte loss without which terrestrial life could not be maintained. Accordingly, acute disruption of the skin through physical or chemical trauma needs to be repaired timely and efficiently as sustained skin pathologies ranging from mild irritations and inflammation through to malignancy impact considerably on morbidity and mortality. The Nuclear Hormone Receptor Family of transcriptional regulators has proven to be highly valuable targets for addressing a range of pathologies, including metabolic syndrome and cancer. Indeed members of the classic endocrine sub-group, such as the glucocorticoid, retinoid, and Vitamin D receptors, represent mainstay treatment strategies for numerous inflammatory skin disorders, though side effects from prolonged use are common. Emerging evidence has now highlighted important functional roles for nuclear receptors belonging to the adopted and orphan subgroups in skin physiology and patho-physiology. This review will focus on these subgroups and explore the current evidence that suggests these nuclear receptor hold great promise as future stand-alone or complementary drug targets in treating common skin diseases and maintaining skin homeostasis.

MC1R and NR4A receptors in cellular stress and DNA repair: implications for UVR protection.

Ultraviolet radiation (UVR) is the most common mutagen that melanocytes are exposed to. UVR causes a diverse range of DNA photolesions contributing to genome instability and promotes melanoma and non-melanoma development. Melanocytes are pigment-producing cells that synthesise the photoprotective melanins when the melanocortin-1 receptor (MC1R) is activated. MC1R is a G-protein-coupled receptor expressed predominantly in melanocytes. Its signalling pathway has been directly linked to melanogenesis, enhanced cytoprotection against UV damage and augmented DNA repair response. Interestingly, previous studies have revealed that MC1R signalling induces the transcription of the NR4A subfamily of orphan nuclear receptors in response to UV. In line with this, studies have also observed that NR4A receptors are recruited to distinct nuclear foci in response to cellular stress, independent of their transcriptional roles. Here, we review the regulated expression of NR4A2 and its potential roles upon cellular stress conditions. Current work in developing synthetic NR4A2 agonists further provides exciting avenues for exploring the potential role of NR4A2 as an antiskin cancer drug target.

The NR4A2 nuclear receptor is recruited to novel nuclear foci in response to UV irradiation and participates in nucleotide excision repair.

Ultraviolet radiation (UVR) is one of the most common mutagens encountered by humans and induces the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproduct (6-4PP) lesions in the genomic DNA. To prevent the accumulation of deleterious mutations these lesions must be efficiently repaired, primarily by nucleotide excision repair. We have previously demonstrated that the NR4A family of nuclear receptors are crucial mediators of the DNA repair function of the MC1R signalling pathway in melanocytes. Here we explore the role of the NR4A2 protein in the DNA repair process further. Using EYFP tagged-NR4A2 we have demonstrated a UVR induced recruitment to distinct nuclear foci where they co-localise with known DNA repair proteins. We reveal that the N-terminal domain of the receptor is required for this translocation and identify a role for p38 and PARP signalling in this process. Moreover disruption of the functional integrity of the Ligand Binding Domain of the receptor by deleting the terminal helix 12 effectively blocks co-localisation of the receptor with DNA repair factors. Restored co-localisation of the mutant receptor with DNA repair proteins in the presence of a Histone Deacetylase Inhibitor suggests that impaired chromatin accessibility underpins the mis-localisation observed. Finally NR4A2 over-expression facilitated a more efficient clearance of UVR induced CPD and 6-4PP lesions. Taken together these data uncover a novel role for the NR4A nuclear receptors as direct facilitators of nucleotide excision repair.

[Studies on chemical constituents of phenols in fresh Houttuynia cordata].

OBJECTIVE: To study the aqueous constituents of Houttuynia cordata. METHOD: Various columns including Diaion HP-20, Sephadex LH-20, ODS and silica gel were employed for the isolation and purification of compounds from H. cordata. The structures of the compounds were identified by physiochemical properties and spectral analysis. RESULT: Five compounds were isolated, and their structures were identified as chlorogenic methyl ester (1), (E)-4-Hydroxy-4-[3'-(beta-D-glucopyranosyloxy) butylidene]-3, 5, 5-trimethyl-2-cyclohexen-1-one (2), 2-(3, 4-dihydroxyphenyl) ethyl-beta-D-glucopyranoside (3), p-hydroxyphenethyl-beta-D-glucoside (4), 4-(beta-D-glucopyranosyloxy)-3-hydroxy-Benzoic acid (5). CONCLUSION: All compounds were isolated from this plant for the first time.

[Study on chemical constituents of flavonoids in fresh herb of Houttuynia cordata].

OBJECTIVE: To study the flavonoid constituents in fresh herb of Houttuynia cordata. METHOD: Various column packing materials including Diaion HP - 20, Sephadex LH - 20, ODS and silica gel were employed for the isolation and purification of compounds from H. cordata. The structures of the compounds were identified by physiochemical properties and spectral analysis. RESULT: Five compounds were isolated, and their structures were identified as quercetin-3-O-beta-D-galactoside-7-O-beta-D-glucoside (1), kaempferol-3-O-alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranoside (2), quercitrin (3), hyperin (4), quercetin 3-O-alpha-L-rhamnopyranosyl-7-O-beta-D-glucopyranoside (5). CONCLUSION: Compounds 1, 2 and 5 were separated from H. cordata for the first time.