Alan Sabirsh

Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes

Summary Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, β, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource.

Leukotriene B4 triggers release of the cathelicidin LL-37 from human neutrophils: novel lipid-peptide interactions in innate immune responses

In humans, the antimicrobial peptide LL‐37 and the potent chemotactic lipid leukotriene B4 (LTB4) are important mediators of innate immunity and host defense. Here we show that LTB4, at very low (1 nM) concentrations, strongly promotes release of LL‐37 peptides from human neutrophils (PMNs) in a time‐ and dose‐dependent manner, as determined by Western blot, enzyme‐linked immunoassay (ELISA), and antibacterial activity. The LTB4‐induced LL‐37 release is mediated by the BLT1 receptor, and protein phosphatase‐1 (PP‐1) inhibits the release by suppressing the BLTl‐mediated exocytosis of PMN granules. Conversely, LL‐37 elicits translocation of 5‐lipoxygen‐ase (5‐LO) from the cytosol to the perinuclear membrane in PMNs and promotes the synthesis and release of LTB4, particularly from cells primed with LPS or GM‐CSF. Furthermore, LL‐37 stimulates PMN phagocytosis of Escherichia coli particles, a functional response that is enhanced by LTB4, especially in GM‐CSF pretreated cells. In these cells, LL‐37 also enhances LTB4‐induced phagocytosis. Hence, in human PMNs, positive feedback circuits exist between LL‐37 and LTB4 that reciprocally stimulate the release of these mediators with the potential for synergistic bioactions and enhanced immune responses. Moreover, these novel lipid‐peptide signaling pathways may offer new opportunities for pharmacological intervention and treatment of chronic inflammatory diseases.—Wan, M., Sabirsh, A., Wetterholm, A., Agerberth, B., Haegg‐ström, J. Z. Leukotriene B4 triggers release of the cathelicidin LL‐37 from human neutrophils: novel lipid‐peptide interactions in innate immune responses. FASEB J. 21, 2897–2905 (2007)

Long-Term Chronic Toxicity Testing Using Human Pluripotent Stem Cell–Derived Hepatocytes

Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.

Pro- and anti-inflammatory substances modulate expression of the leukotriene B4 receptor, BLT1, in human monocytes.

The high‐affinity leukotriene B4 (LTB4) receptor, BLT1, is a chemotactic receptor involved in inflammatory responses. In this study, we have explored the regulation of BLT1 expression in human monocytes by pro‐ and anti‐inflammatory cytokines, lipopolysaccharide (LPS), and dexamethasone. We found that proinflammatory mediators, such as interferon‐γ (IFN‐γ), tumor necrosis factor‐α, and LPS, down‐regulated expression, whereas the anti‐inflammatory cytokine, interleukin‐10, and dexamethasone up‐regulated BLT1 mRNA expression. The effect of IFN‐γ on BLT1 mRNA expression was rapidly detectable (<4 h) and concentration‐dependent (1–50 ng/ml) and seems to be exerted through a block in transcriptional activity. Alterations in mRNA expression were accompanied by changes in BLT1 surface expression, and receptor down‐modulation following IFN‐γ stimulation resulted in a diminished chemotactic response to LTB4. The regulation of BLT1 mRNA and receptor protein expression was similar to the regulation of the monocyte chemoattractant protein‐1 chemokine receptor, CC chemokine recptor 2 (CCR2). Flow cytometric analysis of fresh peripheral blood cells revealed that classical (CD14++CD16–) monocytes express high levels of BLT1 and CCR2 and that both receptors are down‐regulated on CD14+CD16+ monocytes. Apart from providing insight into the regulation of BLT1 in human monocytes, our results reveal a parallel expression and regulation of BLT1 and CCR2, which may help to understand monocyte trafficking during pathophysiological conditions.

15-deoxy-Delta12,14-prostaglandin J2 inhibits the expression of microsomal prostaglandin E synthase type 2 in colon cancer cells.

Prostaglandin (PG) E2 (PGE2) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE2 is accomplished by conversion of the cyclooxygenase (COX) product PGH2 by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Δ12,14-PGJ2 and PGA2 downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor γ or PGD2 receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE2. Our data suggest that there is a feedback mechanism between PGE2 and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.

Inhibition of T-cell activation by the CTLA4-Fc Abatacept is sufficient to ameliorate proteinuric kidney disease

Diabetic nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria, and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.

Impaired Coronary and Renal Vascular Function in Spontaneously Type 2 Diabetic Leptin-Deficient Mice

Background Type 2 diabetes is associated with macro- and microvascular complications in man. Microvascular dysfunction affects both cardiac and renal function and is now recognized as a main driver of cardiovascular mortality and morbidity. However, progression of microvascular dysfunction in experimental models is often obscured by macrovascular pathology and consequently demanding to study. The obese type 2 diabetic leptin-deficient (ob/ob) mouse lacks macrovascular complications, i.e. occlusive atherosclerotic disease, and may therefore be a potential model for microvascular dysfunction. The present study aimed to test the hypothesis that these mice with an insulin resistant phenotype might display microvascular dysfunction in both coronary and renal vascular beds. Methods and Results In this study we used non-invasive Doppler ultrasound imaging to characterize microvascular dysfunction during the progression of diabetes in ob/ob mice. Impaired coronary flow velocity reserve was observed in the ob/ob mice at 16 and 21 weeks of age compared to lean controls. In addition, renal resistivity index as well as pulsatility index was higher in the ob/ob mice at 21 weeks compared to lean controls. Moreover, plasma L-arginine was lower in ob/ob mice, while asymmetric dimethylarginine was unaltered. Furthermore, a decrease in renal vascular density was observed in the ob/ob mice. Conclusion In parallel to previously described metabolic disturbances, the leptin-deficient ob/ob mice also display cardiac and renal microvascular dysfunction. This model may therefore be suitable for translational, mechanistic and interventional studies to improve the understanding of microvascular complications in type 2 diabetes.

Human iPS-Derived Astroglia from a Stable Neural Precursor State Show Improved Functionality Compared with Conventional Astrocytic Models

Summary In vivo studies of human brain cellular function face challenging ethical and practical difficulties. Animal models are typically used but display distinct cellular differences. One specific example is astrocytes, recently recognized for contribution to neurological diseases and a link to the genetic risk factor apolipoprotein E (APOE). Current astrocytic in vitro models are questioned for lack of biological characterization. Here, we report human induced pluripotent stem cell (hiPSC)-derived astroglia (NES-Astro) developed under defined conditions through long-term neuroepithelial-like stem (ltNES) cells. We characterized NES-Astro and astrocytic models from primary sources, astrocytoma (CCF-STTG1), and hiPSCs through transcriptomics, proteomics, glutamate uptake, inflammatory competence, calcium signaling response, and APOE secretion. Finally, we assess modulation of astrocyte biology using APOE-annotated compounds, confirming hits of the cholesterol biosynthesis pathway in adult and hiPSC-derived astrocytes. Our data show large diversity among astrocytic models and emphasize a cellular context when studying astrocyte biology.

Long noncoding RNAs are dynamically regulated during β-cell mass expansion in mouse pregnancy and control β-cell proliferation in vitro

Pregnancy is associated with increased β-cell proliferation driven by prolactin. Long noncoding RNAs (lncRNA) are the most abundant RNA species in the mammalian genome, yet, their functional importance is mainly elusive. Aims/hypothesis: This study tests the hypothesis that lncRNAs regulate β-cell proliferation in response to prolactin in the context of β-cell mass compensation in pregnancy. Methods: The expression profile of lncRNAs in mouse islets at day 14.5 of pregnancy was explored by a bioinformatics approach, further confirmed by quantitative PCR at different days of pregnancy, and islet specificity was evaluated by comparing expression in islets versus other tissues. In order to establish the role of the candidate lncRNAs we studied cell proliferation in mouse islets and the MIN6 β-cell line by EdU incorporation and cell count. Results: We found that a group of lncRNAs is differentially regulated in mouse islets at 14.5 days of pregnancy. At different stages of pregnancy, these lncRNAs are dynamically expressed, and expression is prolactin dependent in mouse islets and MIN6 cells. One of those lncRNAs, Gm16308 (Lnc03), is dynamically regulated during pregnancy, prolactin-dependent and islet-enriched. Silencing Lnc03 in primary β-cells and MIN6 cells inhibits, whereas over-expression stimulates, proliferation even in the absence of prolactin, demonstrating that Lnc03 regulates β-cell growth. Conclusions/interpretation: During pregnancy mouse islet proliferation is correlated with dynamic changes of lncRNA expression. In particular, Lnc03 regulates mouse β-cell proliferation and may be a crucial component of β-cell proliferation in β-cell mass adaptation in both health and disease.

Reduced Baseline Sensitivity to Maraviroc Inhibition Among R5 HIV-1 Isolates From Individuals With Severe Immunodeficiency

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