Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils

Background

Subepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.

Objective

In this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.

Methods

Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.

Results

The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.

Conclusions

Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases.     

Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.     

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5′-, but not 3′-ends of biotinylated DNA.     

Cyclin-Dependent Kinase Five Mediates Activation of Lung Xanthine Oxidoreductase in Response to Hypoxia

Background

Xanthine oxidoreductase (XOR) is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS). As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s) responsible for such hyperactivation are unknown.

Methods and Results

Using an in silico approach, we identified a cyclin-dependent kinase 5 (CDK5) consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD) binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC) and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25), and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222) within the CDK5-consensus motif.

Conclusions and Significance

These results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury.     

Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS

Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.     

Involvement of neuroleptic drugs in selenium deficiency and sudden death of cardiac origin: study and human post-mortem examination

The involvement of psychotropic drugs in sudden deaths has been highlighted. The objective of this work was to establish a link between selenium levels in heart tissue, psychotropic treatment and sudden death. Selenium levels were measured by electrothermal atomic absorption spectroscopy post-mortem in heart, brain and liver. Histological examination evidenced dilated cardiomyopathy in 45% of cases, left ventricular hypertrophy in 36%, and ischemic coronaropathy in 18%. A significant reduction of myocardial selenium levels compared to controls was seen in patients treated with neuroleptic drugs or meprobamate. No changes in brain or liver selenium levels were seen. These results suggest that selenium deficiency can facilitate sudden death in patients on psychotropic drugs. The reduced activity of glutathione peroxidase due to selenium deficiency can result in augmented oxidative stress in myocardial cells and myocardiopathy leading to sudden death.     

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.     

High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.     

Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M(3) muscarinic acetylcholine receptor

To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.