A liver immune rheostat regulates CD8 T cell immunity in chronic HBV infection

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3–7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12–22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP–PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase–cAMP–PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase–cAMP–PKA axis in an immune rheostat-like fashion.</p

Use of toxicogenomics in drug safety evaluation: Current status and an industry perspective

Toxicogenomics held great promise as an approach to enable early detection of toxicities induced by xenobiotics; however, there remain questions regarding the impact of the discipline on pharmaceutical nonclinical safety assessment. To understand the current state of toxicogenomics in the sector, an industry group surveyed companies to determine the frequency of toxicogenomics use in in vivo studies at various stages of drug discovery and development and to assess how toxicogenomics use has evolved over time. Survey data were compiled during 2016 from thirteen pharmaceutical companies. Toxicogenomic analyses were infrequently conducted in the development phase and when performed were done to address specific mechanistic questions. Prior to development, toxicogenomics use was more frequent; however, there were significant differences in approaches among companies. Across all phases, gaining mechanistic insight was the most frequent reason cited for pursing toxicogenomics with few companies using toxicogenomics to predict toxicities. These data were consistent with the commentary submitted in response to survey questions asking companies to describe the evolution of their toxicogenomics strategy. Overall, these survey data indicate that toxicogenomics is not widely used as a predictive tool in the pharmaceutical industry but is used regularly by some companies and serves a broader role in mechanistic investigations and as a complement to other technologies

Cardiac spheroids as promising in vitro models to study the human heart microenvironment

Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart (“cardiac spheroids”, CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue

Epithelial cell injury characterization (upper panel) and fibroblast activation (lower panel) in an <i>in vitro</i> reconstructed microenvironment.

<p>(<b>A</b>) Scheme of the reconstructed microenvironment and workflow analysis of the cisplatin-injured proximal tubular epithelial cells HKC-8 cells and of the WS-1 dermal fibroblasts. (<b>B</b>) Cell viability and (<b>C</b>) apoptosis analysis. Cisplatin-treated proximal tubular epithelial cells HKC-8 cells showed decreased cell viability and increased apoptosis. (<b>D</b>). Cell cycle analysis showed that HKC-8 cells treated with cisplatin high dose (40 µM) were blocked in G2/M phase at 24, 48 and 72 h, whereas cells treated with the low dose (20 µM) reverted at 72 h to a condition similar to control. Cytokine release analysis with (<b>E</b>) IL-6 and (<b>F</b>) RANTES levels. Cisplatin-treated HKC-8 cells produced increased amounts of IL-6 and RANTES. (<b>G</b>) Gene-level analysis results for selected genes showing a stronger response to Ciplatin high dose (CisHigh) than to Ciplatin low dose (CisLow). Expression levels on a logarithmic scale are shown as a heat map: no detectable expression is indicated by black color, increasing expression levels are indicated by brighter shades of yellow. Note that several genes show up twice in the figure because they are represented by multiple probes on the Illumina chip. While the measured values do not necessarily agree, the overall trend of up-regulation is the same. (<b>H</b>) Gene-level analysis was complemented by a network-level approach using Gene Set Enrichment Analysis against the Pathway Commons collection of gene regulatory networks (<a href="http://www.pathwaycommons.org" target="_blank">www.pathwaycommons.org</a>). Cisplatin treated cells (L: low, H: high) were compared to controls (C), and renal clear cell carcinoma (RCC) cells were compared to “normal adjacent” tissue (GEO accession number GSE781; as this data set is based on a different expression array technology, we did not compare expression levels of individual genes for this analysis). The heat map shows FDR-corrected q values on a logarithmic scale for up-regulated (red shades) and down-regulated networks (green shades), black indicating no change. An FDR-corrected q value of 0.01 corresponds to an absolute score of 4.6 on this scale. Please, note that the RCC dataset (last column) does not imply any involvement of the networks shown here. (<b>I–L</b>) RT-PCR analysis and mRNA levels of the (<b>I</b>) <i>Acta2</i> gene (encoding alpha smooth muscle actin) (<b>J</b>) <i>TGF-b1</i>gene (encoding transforming growth factor beta 1), (<b>K</b>) <i>COL1A1</i> gene (encoding collagen-1α1) and (<b>L</b>) ID-1 gene (encoding Inhibitor of differentiation 1). Retrieved WS-1 dermal fibroblasts showed increased level for key fibrotic markers α-SMA, TGF-β1 and Collagen 1α1 and decreased level of ID-1 when epithelial cells HK-C8 cells were layered on top. Gene expression profile for the same gene in absence of HK-C8 cells can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056575#pone.0056575.s002" target="_blank">Figure S2B</a>-E. n.s. = not statistically different, * = p<0.05, ** = p<0.001.</p

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator

ISSN:1096-6080ISSN:1096-092

STAT3 Gain of Function: A New Kid on the Block in Interstitial Lung Diseases

International audienceA 5-year-old girl with failure to thrive and multiorgan disease was referred to our center for chronic hypoxemia. On evaluation, we noted tachypnea (respiratory rate 35/min), supraclavicular retractions, median diurnal oxygen saturation as measured by pulse oximetry (Sp O 2) = 91.7% at rest, percentage of time below Sp O 2 90% at 26% during sleep, and clubbing. A computed tomography scan showed diffuse interstitial lung disease (Figure 1). Spirometry was normal (TLC, 83% of predicted; FEV 1 , 83% of predicted; FEV 1 /FVC, 98%; and forced expiratory flow, midexpiratory phase, 142% of predicted), but it was not possible to measure DL CO

Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance