Probes for INS

In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3-6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI.

Abstract

BACKGROUND:

Progressive familial intrahepatic cholestasis type 3 (PFIC3) often leads to end-stage liver disease before adulthood with limited therapeutic options, due to impaired ABCB4 dependent phospholipid transport to bile. To restore ABCB4 function we propose adeno-associated virus serotype 8 (AAV8)-mediated gene therapy directed to the liver, although achieving stable transgene expression in hyperproliferative tissue is challenging. By restoring the phospholipid content in bile to levels that prevent liver damage, this study aims for stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3.

METHODS:

Ten weeks old Abcb4-/- mice received a single dose of AAV8-hABCB4 (n=10) or AAV8-GFP (n=7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by (immuno-)histochemistry.

RESULTS:

Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration, which restored biliary phospholipid excretion to levels that ameliorate liver damage. This resulted in normalization of plasma cholestatic markers, prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study.

CONCLUSION:

Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3, encouraging translational studies to verify clinical feasibility.

LAY SUMMARY:

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. Also proliferation of liver cells was reduced, which contributes to long term correction of the phenotype. Limitations of the mouse model requires further studies to evaluate if this approach can be applied in PFIC3 patients.

The HSV1 virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in shut-off of host protein synthesis. Hence its unrestrained activity is considered to be lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from co-transfected plasmids were also retained in the same nuclei where vhs mRNA was located, while polyA binding protein (PABP) was relocalised to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Co-expression of VP16 and VP22 rescued cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5' region of vhs that blocked its translation and, when transferred to a heterologous GFP transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and auto-induced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.IMPORTANCE A myriad of gene expression strategies has been discovered through studies carried out on viruses. This report concerns the regulation of the HSV1 vhs endoribonuclease, a virus factor that is important for counteracting host antiviral responses by degrading their mRNAs, but which must be regulated during infection to ensure that it does not act against and inhibit the virus itself. We show that regulation of vhs involves multifaceted post-transcriptional cellular and viral processes, including aberrant mRNA localization and a novel, autoregulated negative feedback loop to target its own and co-expressed mRNAs for nuclear retention, an activity that is relieved by co-expression of two other virus proteins, VP22 and VP16. These studies reveal the interplay of strategies by which multiple virus-encoded factors co-ordinate gene expression at the time they are needed. These findings are broadly relevant to both virus and cellular gene expression.

Pancreatic islet cells are critical for maintaining normal blood glucose levels, and their malfunction underlies diabetes development and progression. We used single-cell RNA sequencing to determine the transcriptomes of 1,492 human pancreatic α, β, δ, and PP cells from non-diabetic and type 2 diabetes organ donors. We identified cell-type-specific genes and pathways as well as 245 genes with disturbed expression in type 2 diabetes. Importantly, 92% of the genes have not previously been associated with islet cell function or growth. Comparison of gene profiles in mouse and human α and β cells revealed species-specific expression. All data are available for online browsing and download and will hopefully serve as a resource for the islet research community.

The ventral pallidum (VP) lies at the interface between sensory, motor, and cognitive processing-with a particular role in mounting behavioral responses to rewards. Though the VP is predominantly GABAergic, glutamate neurons were recently identified, though their relative abundances and respective roles are unknown. Here, we show that VP glutamate neurons are concentrated in the rostral ventromedial VP and project to qualitatively similar targets as do VP GABA neurons. At the functional level, we used optogenetics to show that activity in VP GABA neurons can drive positive reinforcement, particularly through projections to the ventral tegmental area (VTA). On the other hand, activation of VP glutamate neurons leads to behavioral avoidance, particularly through projections to the lateral habenula. These findings highlight cell-type and projection-target specific roles for VP neurons in behavioral reinforcement, dysregulation of which could contribute to the emergence of negative symptoms associated with drug addiction and other neuropsychiatric disease.