NOD Signaling and Cell Death

Innate immune signaling and programmed cell death are intimately linked, and many signaling pathways can regulate and induce both, transcription of inflammatory mediators or autonomous cell death. The best-characterized examples for these dual outcomes are members of the TNF superfamily, the inflammasome receptors, and the toll-like receptors. Signaling via the intracellular peptidoglycan receptors NOD1 and NOD2, however, does not appear to follow this trend, despite involving signaling proteins, or proteins with domains that are linked to programmed cell death, such as RIP kinases, inhibitors of apoptosis (IAP) proteins or the CARD domains on NOD1/2. To better understand the connections between NOD signaling and cell death induction, we here review the latest findings on the molecular regulation of signaling downstream of the NOD receptors and explore the links between this immune signaling pathway and the regulation of cell death

Posttranscriptional regulation of Fas (CD95) ligand killing activity by lipid rafts

Fas (CD95/Apo-1) ligand-mediated apoptosis induction of target cells is one of the major effector mechanisms by which cytotoxic lymphocytes (T cells and natural killer cells) kill their target cells. In T cells, Fas ligand expression is tightly regulated at a transcriptional level through the activation of a distinct set of transcription factors. Increasing evidence, however, supports an important role for posttranscriptional regulation of Fas ligand expression and activity. Lipid rafts are cholesterol- and sphingolipid-rich membrane microdomains, critically involved in the regulation of membrane receptor signaling complexes through the clustering and concentration of signaling molecules. Here, we now provide evidence that Fas ligand is constitutively localized in lipid rafts of FasL transfectants and primary T cells. Importantly, disruption of lipid rafts strongly reduces the apoptosis-inducing activity of Fas ligand. Localization to lipid rafts appears to be predominantly mediated by the characteristic cytoplasmic proline-rich domain of Fas ligand because mutations of this domain result in reduced recruitment to lipid rafts and attenuated Fas ligand killing activity. We conclude that Fas ligand clustering in lipid rafts represents an important control mechanism in the regulation of T cell-mediated cytotoxicity

IAPs Regulate Distinct Innate Immune Pathways to Co-ordinate the Response to Bacterial Peptidoglycans

Summary: Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling in vivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways

The combination of tacrolimus and MMF promotes cell death in hepatocytes.

<p>(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.005 μg/ml of tacrolimus ± 5 μg/ml of MMF or 1 μg/ml of cyclosporine + 5 μg/ml of MMF at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.005 μg/ml of tacrolimus ± 5 μg/ml of MMF or 1 μg/ml of cyclosporine + 5 μg/ml of MMF at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in tacrolimus/MMF-treated PMoH and cyclosporine/MMF-treated cells compared to untreated hepatocytes. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.</p

The combination of sirolimus and mycophenolate mofetil has no significant effect on cell death in hepatocytes.

<p>(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.01 μg/ml of sirolimus ± 5 μg/ml of MMF or 0.005 μg/ml of tacrolimus + 5 μg/ml of MMF at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.01 μg/ml of sirolimus ± 5 μg/ml of MMF or 0.005 μg/ml of tacrolimus + 5 μg/ml of MMF at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in sirolimus/MMF-treated PMoH and tacrolimus/MMF-treated cells compared to untreated hepatocytes. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.</p

Tacrolimus at therapeutic concentrations does not affect hepatocyte cell death.

<p>(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.005 μg/ml of tacrolimus at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.005 μg/ml of tacrolimus at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in tacrolimus-treated PMoH relative to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.</p

Sirolimus at therapeutic concentrations does not increase hepatocyte cell death.

<p>(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 0.01 μg/ml of sirolimus at 24–72 hours compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 0.01 μg/ml of sirolimus at 48 hours compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels in sirolimus-treated PMoH compared to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.</p

Patient demographics and clinical data for the 10 post-liver transplant patients and 9 control patients whose liver sections were analyzed for markers of apoptosis.

<p>Post-Tx: Post-liver transplant; Bx: Liver biopsy; Alb: Serum albumin; Bili: Serum bilirubin; ALT: Serum alanine transaminase; INR: International Normalized Ratio; CyA: Cyclosporine; Aza: Azathioprine; Tac: Tacrolimus; Pred: Prednisolone; MMF: Mycophenolate mofetil.</p><p>Patient demographics and clinical data for the 10 post-liver transplant patients and 9 control patients whose liver sections were analyzed for markers of apoptosis.</p

Cyclosporine at therapeutic concentrations does not increase hepatocyte cell death.

<p>(A) Percentage reduction in cell viability from crystal violet assays of PMoH treated with 1 μg/ml of cyclosporine compared to untreated cells. (B) Western blots of cleaved PARP and cleaved caspase 3 levels in PMoH treated with 1 μg/ml of cyclosporine compared to untreated cells. Graphs show fold change in cleaved PARP and cleaved caspase 3 levels relative to untreated cells. Each bar represents the average of 3 experiments and error bar represents SEM. P-values are compared to untreated hepatocytes.</p