Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia.

Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5'-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy

IL-17 Expression in the Time Course of Acute Anti-Thy1 Glomerulonephritis

Background Interleukin-17 (IL-17) is a new pro-inflammatory cytokine involved in immune response and inflammatory disease. The main source of IL-17 is a subset of CD4+ T-helper cells, but is also secreted by non-immune cells. The present study analyzes expression of IL-17 in the time course of acute anti- thy1 glomerulonephritis and the role of IL-17 as a potential link between inflammation and fibrosis. Methods Anti-thy1 glomerulonephritis was induced into male Wistar rats by OX-7 antibody injection. After that, samples were taken on days 1, 5, 10 (matrix expansion phase), 15 and 20 (resolution phase). PBS-injected animals served as controls. Proteinuria and histological matrixes score served as the main markers for disease severity. In in vitro experiments, NRK-52E cells were used. For cytokine expressions, mRNA and protein levels were analyzed by utilizing RT-PCR, in situ hybridization and immunofluorescence. Results Highest IL-17 mRNA-expression (6.50-fold vs. con; p<0.05) was found on day 5 after induction of anti-thy1 glomerulonephritis along the maximum levels of proteinuria (113 ± 13 mg/d; p<0.001), histological glomerular-matrix accumulation (82%; p<0.001) and TGF-β1 (2.2-fold; p<0.05), IL-6 mRNA expression (36-fold; p<0.05). IL-17 protein expression co-localized with the endothelial cell marker PECAM in immunofluorescence. In NRK-52E cells, co-administration of TGF-β1 and IL-6 synergistically up-regulated IL-17 mRNA 4986-fold (p<0.001). Conclusions The pro-inflammatory cytokine IL-17 is up-regulated in endothelial cells during the time course of acute anti-thy1 glomerulonephritis. In vitro, NRK-52E cells secrete IL-17 under pro-fibrotic and pro-inflammatory conditions

Pathological RANK signaling in B cells drives autoimmunity and chronic lymphocytic leukemia

Clinical evidence suggests alterations in receptor activator of NF-κB (RANK) signaling are key contributors to B cell autoimmunity and malignancy, but the pathophysiological consequences of aberrant B cell-intrinsic RANK signaling remain unknown. We generated mice that express a human lymphoma-derived, hyperactive RANKK240E variant in B lymphocytes in vivo. Forced RANK signaling disrupted B cell tolerance and induced a fully penetrant systemic lupus erythematosus-like disease in addition to the development of chronic lymphocytic leukemia (CLL). Importantly, RANKK240E transgenic CLL cells as well as CLL cells of independent murine and of human origin depend on microenvironmental RANK ligand (RANKL) for tumor cell survival. Consequently, inhibition of the RANKL-RANK axis with anti-RANKL antibodies killed murine and human CLL cells in vitro and in vivo. These results establish pathological B cell-intrinsic RANK signaling as a potential driver of autoimmunity and B cell malignancy, and they suggest the exploitation of clinically available anti-RANKL compounds for CLL treatment

Expression and activity of soluble guanylate cyclase in injury and repair of anti-thy1 glomerulonephritis

Expression and activity of soluble guanylate cyclase in injury and repair of anti-thy1 glomerulonephritis.BackgroundActivation of soluble guanylate cyclase and generation of cyclic 3′,5′-guanosine monophosphate (cGMP) is the main signal transducing event of the L-arginine-nitric oxide pathway. The present study analyzes the expression and activity of the nitric oxide-cGMP signaling cascade in and the effect of the specific soluble guanylate cyclase stimulator Bay 41-2272 on the early injury and subsequent repair phase of acute anti-thy1 glomerulonephritis.MethodsAnti-thy1 glomerulonephritis was induced by OX-7 antibody injection in rats. In protocol 1 (injury), Bay 41-2272 was given starting 6 days before antibody injection. One day after disease induction, parameters of mesangial cell injury (glomerular cell number and inducible nitric oxide synthesis) were analyzed. In protocol 2 (repair), Bay 41-2272 treatment was started one day after antibody injection. On day 7, parameters of glomerular repair [glomerular matrix score, expression of transforming growth factor (TGF)-β1, fibronectin, and plasminogen-activator-inhibitor (PAI)-1, infiltration with macrophages and fibrinogen deposition (indicating platelet localization)] were determined. In both protocols, tail bleeding time, systolic blood pressure, plasma cGMP levels, glomerular mRNA expression of endothelial nitric oxide synthase (eNOS), α1 and β1 soluble guanylate cyclase, and basal and nitric oxide-stimulated glomerular cGMP production were analyzed.ResultsBay 41-2272 prolonged bleeding time, reduced blood pressure, and increased plasma cGMP levels in both protocols. In the injury experiment, disease induction increased inducible nitric oxide synthesis and reduced glomerular cell number, while expression and activity of soluble guanylate cyclase was almost completely diminished. Bay 41-2272 did not affect parameters of mesangial cell injury and glomerular soluble guanylate cyclase expression and activity. In the repair protocol, expression and activity of soluble guanylate cyclase was markedly increased by disease. Bay 41-2272 further enhanced soluble guanylate cyclase expression and activity. This went along with significant reductions in proteinuria, glomerular matrix accumulation, expression of TGF-β1, fibronectin, and PAI-1, macrophage infiltration and fibrinogen deposition as compared to the untreated anti-thy1 animals.ConclusionGlomerular nitric oxide signaling via cGMP is markedly impaired during injury of anti-thy1 glomerulonephritis, while it is highly up-regulated during subsequent repair. Further pharmacologic soluble guanylate cyclase stimulation limits glomerular TGF-β overexpression and matrix expansion, suggesting that the soluble guanylate cyclase enzyme represents an important antifibrotic pathway in glomerular disease

TGF-β1, IL-6 and IL-17 mRNA expression of NRK-52E after glucose, IL-6, TGF-β1 or IL-17 stimulation.

<p>TGF-β1, IL-6 and IL-17 mRNA expression in NRK-52E cells 15 min after stimulation by 25 mM glucose, 10 ng/ml IL-6, 5 ng/ml TGF-β1 or 25 ng/ml IL-17. Analysis utilized real-time PCR and was normalized to β-actin as housekeeping gene. (# p <0.05 vs. nc; * p<0.01 vs. nc).</p

IL-17 and PECAM-1 protein expression of an anti-thy1 glomerulonephritic animal on d5.

<p>Representative images showing glomerular IL-17 and PECAM-1 protein expression in renal tissue sections of an anti-thy1 glomerulonephritic animal on d5 after disease induction, in paraffin-embedded immunostained sections at x400 magnification. IL-17 green, PCAM-1 red, nucleus blue, merge yellow.</p

IL-17 and synaptopodin protein expression of an anti-thy1 glomerulonephritic animal on d5.

<p>Representative images showing glomerular IL-17 and synaptopodin protein expression in renal tissue sections of an anti-thy1 glomerulonephritic animal on d5 after disease induction, in paraffin-embedded immunostained sections at x400 magnification. IL-17 green, synaptopodin red, nucleus blue, merge yellow.</p