Oxidant Signaling Mediated by Nox2 in Neutrophils Promotes Regenerative Myelopoiesis and Tissue Recovery following Ischemic Damage.

Ischemic tissue damage activates hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM)-generating myeloid cells, and persistent HSPC activity may drive chronic inflammation and impair tissue recovery. Although increased reactive oxygen species in the BM regulate HSPC functions, their roles in myelopoiesis of activated HSPCs and subsequent tissue recovery during ischemic damage are not well understood. In this paper, we report that deletion of Nox2 NADPH oxidase in mice results in persistent elevations in BM HSPC activity and levels of inflammatory monocytes/macrophages in BM and ischemic tissue in a model of hindlimb ischemia. Ischemic tissue damage induces oxidants in BM such as elevations of hydrogen peroxide and oxidized phospholipids, which activate redox-sensitive Lyn kinase in a Nox2-dependent manner. Moreover, during tissue recovery after ischemic injury, this Nox2-ROS-Lyn kinase axis is induced by Nox2 in neutrophils that home to the BM, which inhibits HSPC activity and inflammatory monocyte generation and promotes tissue regeneration after ischemic damage. Thus, oxidant signaling in the BM mediated by Nox2 in neutrophils regulates myelopoiesis of HSPCs to promote regeneration of damaged tissue.

Deleted in Breast Cancer 1 Suppresses B Cell Activation through RelB and Is Regulated by IKKα Phosphorylation.

Alternative NF-κB signaling is crucial for B cell activation and Ig production, and it is mainly regulated by the inhibitor of κ B kinase (IKK) regulatory complex. Dysregulation of alternative NF-κB signaling in B cells could therefore lead to hyperactive B cells and Ig overproduction. In our previous, study we found that deleted in breast cancer 1 (DBC1) is a suppressor of the alternative NF-κB pathway to attenuate B cell activation. In this study, we report that loss of DBC1 results in spontaneous overproduction of Ig in mice after 10 mo of age. Using a double mutant genetic model, we confirm that DBC1 suppresses B cell activation through RelB inhibition. At the molecular level, we show that DBC1 interacts with alternative NF-κB members RelB and p52 through its leucine zipper domain. In addition, phosphorylation of DBC1 at its C terminus by IKKα facilitates its interaction with RelB and IKKα, indicating that DBC1-mediated suppression of alternative NF-κB is regulated by IKKα. Our results define the molecular mechanism of DBC1 inhibition of alternative NF-κB activation in suppressing B cell activation.

DBC1 is a suppressor of B cell activation by negatively regulating alternative NF-κB transcriptional activity.

CD40 and BAFFR signaling play important roles in B cell proliferation and Ig production. In this study, we found that B cells from mice with deletion of Dbc1 gene (Dbc1(-/-)) show elevated proliferation, and IgG1 and IgA production upon in vitro CD40 and BAFF, but not BCR and LPS stimulation, indicating that DBC1 inhibits CD40/BAFF-mediated B cell activation in a cell-intrinsic manner. Microarray analysis and chromatin immunoprecipitation experiments reveal that DBC1 inhibits B cell function by selectively suppressing the transcriptional activity of alternative NF-κB members RelB and p52 upon CD40 stimulation. As a result, when immunized with nitrophenylated-keyhole limpet hemocyanin, Dbc1(-/-) mice produce significantly increased levels of germinal center B cells, plasma cells, and Ag-specific Ig. Finally, loss of DBC1 in mice leads to higher susceptibility to experimental autoimmune myasthenia gravis. Our study identifies DBC1 as a novel regulator of B cell activation by suppressing the alternative NF-κB pathway.

GM-CSF: An immune modulatory cytokine that can suppress autoimmunity.

GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases such as Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance.

Recombinant IgG2a Fc (M045) multimers effectively suppress experimental autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disorder caused by target-specific pathogenic antibodies directed toward postsynaptic neuromuscular junction (NMJ) proteins, most commonly the skeletal muscle nicotinic acetylcholine receptor (AChR). In MG, high-affinity anti-AChR Abs binding to the NMJ lead to loss of functional AChRs, culminating in neuromuscular transmission failure and myasthenic symptoms. Intravenous immune globulin (IVIg) has broad therapeutic application in the treatment of a range of autoimmune diseases, including MG, although its mechanism of action is not clear. Recently, the anti-inflammatory and anti-autoimmune activities of IVIg have been attributed to the IgG Fc domains. Soluble immune aggregates bearing intact Fc fragments have been shown to be effective treatment for a number of autoimmune disorders in mice, and fully recombinant multimeric Fc molecules have been shown to be effective in treating collagen-induced arthritis, murine immune thrombocytopenic purpura, and experimental inflammatory neuritis. In this study, a murine model of MG (EAMG) was used to study the effectiveness of this novel recombinant polyvalent IgG2a Fc (M045) in treating established myasthenia, with a direct comparison to treatment with IVIg. M045 treatment had profound effects on the clinical course of EAMG, accompanied by down-modulation of pathogenic antibody responses. These effects were associated with reduced B cell activation and T cell proliferative responses to AChR, an expansion in the population of FoxP3(+) regulatory T cells, and enhanced production of suppressive cytokines, such as IL-10. Treatment was at least as effective as IVIg in suppressing EAMG, even at doses 25-30 fold lower. Multimeric Fc molecules offer the advantages of being recombinant, homogenous, available in unlimited quantity, free of risk from infection and effective at significantly reduced protein loads, and may represent a viable therapeutic alternative to polyclonal IVIg.

Impaired regulatory function in circulating CD4(+)CD25(high)CD127(low/-) T cells in patients with myasthenia gravis.

Previous studies have reported alterations in numbers or function of regulatory T (Treg) cells in myasthenia gravis (MG) patients, but published results have been inconsistent, likely due to the isolation of heterogenous "Treg" populations. In this study, we used surface CD4, CD25(high), and CD127(low/-) expression to isolate a relatively pure population of Tregs, and established that there was no alteration in the relative numbers of Tregs within the peripheral T cell pool in MG patients. In vitro proliferation assays, however, demonstrated that Treg-mediated suppression of responder T (Tresp) cells was impaired in MG patients and was associated with a reduced expression of FOXP3 in isolated Tregs. Suppression of both polyclonal and AChR-activated Tresp cells from MG patients could be restored using Tregs isolated from healthy controls, indicating that the defect in immune regulation in MG is primarily localized to isolated Treg cells, and revealing a potential novel therapeutic target.

Granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: effects on regulatory T cells.

INTRODUCTION: In this study we describe a patient with a prolonged myasthenic crisis refractory to conventional immunomodulatory therapy who was treated with GM-CSF (granulocyte macrophage colony-stimulating factor, sargramostim). METHODS: T-regulatory cell (Treg) suppressive function and Foxp3 expression were evaluated before and after treatment with GM-CSF. RESULTS: Treatment with GM-CSF was associated with clinical improvement, expansion in the circulating numbers of Foxp3(+) cells, increase in Foxp3 expression levels in Tregs, early improvement in Treg suppressive capacity for AChR-α-induced T-cell proliferation, and subsequent enhancement in Treg suppression of polyclonal T-cell proliferation. CONCLUSION: Although definitive conclusions cannot be drawn from a single case, the correlation with similar findings in GM-CSF-treated animals with experimental autoimmune myasthenia gravis suggests further exploration of the effects of GM-CSF in myasthenia gravis should be studied in a clinical trial setting.

Antioxidant potential of biflavonoid attenuates hyperglycemia by modulating the carbohydrate metabolic enzymes in high fat diet/streptozotocin induced diabetic rats.

Objectives: The present study was to isolate the biflavonoid (a bimolecular kaemferol structured molecule) and test its efficacy on oxidative stress and carbohydrate metabolic key enzymes in control and high fat diet and streptozotocin -induced diabetic rats.Methods: Type 2 diabetes was induced in male albino wistar rats by feeding them with high fat diet comprising of 84.3% standard laboratory chow, 5% lard, 10% yolk powder, cholesterol 0.2%, and 0.5% bile salt for 2 weeks. After 2 weeks, the animals were kept in an overnight fast and injected with low dose of streptozotocin (35 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.5).Results: At the end of the experimental period, diabetic control rats showed significant increase in plasma glucose, homeostatic model assessment of insulin resistance (HOMA-IR), glycosylated hemoglobin (HbA1c) with concomitant decrease in plasma insulin, total hemoglobin and body weight. The activities of key enzymes of carbohydrate metabolism, lipid peroxidation markers, antioxidant enzymes, glycogen content and glycogen synthase and glycogen phosphorylase were also altered in diabetic rats.Discussion: Oral administration of biflavonoid to diabetic rats significantly ameliorated all the biochemical alterations to near normal levels. The effect produced by the biflavonoid on various parameters was comparable to that of metformin.

Effect of ß-sitosterol on glucose homeostasis by sensitization of insulin resistance via enhanced protein expression of PPRγ and glucose transporter 4 in high fat diet and streptozotocin-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: ß-Sitosterol is a plant derived compound similar to cholesterol structure and used in the treatment of hypercholesterolemia, prostate cancer, breast cancer and coronary artery disease. But no studies have been reported the effect of ß-sitosterol on glucose homeostasis by sensitization of insulin resistance via enhanced protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and glucose transporter 4 (GLUT4) in insulin dependent tissues of high fat diet and streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Type 2 diabetes was induced in male albino Wistar rats by feeding them with high fat diet comprising of 84.3% standard laboratory chow, 5% lard, 10% yolk powder, 0.2% cholesterol and 0.5% bile salt for 2 weeks. After 2 weeks, the animals were kept in an overnight fast and injected with low dose of streptozotocin (35 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.5). Analysis of blood glucose, insulin, hemoglobin and glycated hemoglobin were done by commercially available diagnostic kits. The PPARγ and GLUT4 were analyzed by western blotting using respective primary and secondary antibodies. RESULTS: Upon administration of ß-sitosterol at a dose of 15 mg/kg body weight per day to high fat diet and streptozotocin induced diabetic rats for 30 days significantly decreased the levels of plasma glucose, homeostatic model assessment of insulin resistance and glycosylated hemoglobin and increased the levels of insulin, hemoglobin and protein expression of PPARγ and GLUT4 in insulin dependent tissues. Furthermore, ß-sitosterol administration prevented the body weight loss and excessive intake of food and water. CONCLUSION: These finding suggest that ß-sitosterol can replace the commercial drugs which could lead to reduction in toxicity and side effect caused by the later as well as reduce the secondary complications.

Pharmacological inhibition of LSD1 and mTOR reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell disease.

Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders hemoglobin susceptible to polymerization when deoxygenated, affects millions of people worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors eliminate their mitochondria during the terminal differentiation process. Strikingly, we observed an increased percentage of RBCs retaining mitochondria in SCD patient blood samples compared with healthy individuals. In addition, using an experimental SCD mouse model, we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mitochondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1 showed increased expression of mitophagy genes. Our findings suggest that reduction of mitochondria-retaining RBCs may provide a new therapeutic approach to preventing excessive ROS in SCD.

Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy.

Granulocyte macrophage colony stimulating factor (GM-CSF) is generally recognized as an inflammatory cytokine. Its inflammatory activity is primarily due its role as a growth and differentiation factor for granulocyte and macrophage populations. In this capacity, among other clinical applications, it has been used to bolster anti-tumor immune responses. GM-CSF-mediated inflammation has also been implicated in certain types of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Thus, agents that can block GM-CSF or its receptor have been used as anti-inflammatory therapies. However, a review of literature reveals that in many situations GM-CSF can act as an anti-inflammatory/regulatory cytokine. We and others have shown that GM-CSF can modulate dendritic cell differentiation to render them "tolerogenic," which, in turn, can increase regulatory T-cell numbers and function. Therefore, the pro-inflammatory and regulatory effects of GM-CSF appear to depend on the dose and the presence of other relevant cytokines in the context of an immune response. A thorough understanding of the various immunomodulatory effects of GM-CSF will facilitate more appropriate use and thus further enhance its clinical utility.

Functional defect in regulatory T cells in myasthenia gravis.

Forkhead box P3 (FOXP3) is a transcription factor necessary for the function of regulatory T cells (T(reg) cells). T(reg) cells maintain immune homeostasis and self-tolerance and play an important role in the prevention of autoimmune disease. Here, we discuss the role of T(reg) cells in the pathogenesis of myasthenia gravis (MG) and review evidence indicating that a significant defect in T(reg) cell in vitro suppressive function exists in MG patients, without an alteration in circulating frequency. This functional defect is associated with a reduced expression of key functional molecules, such as FOXP3 on isolated T(reg) cells, and appears to be more pronounced in immunosuppression-naive MG patients. In vitro administration of granulocyte macrophage-colony-stimulating factor (GM-CSF) enhanced the suppressive function of T(reg) cells and upregulated FOXP3 expression. These findings indicate a clinically relevant T(reg) cell-intrinsic defect in immune regulation in MG that may reveal a novel therapeutic target.