Nebojsa Arsenijevic

Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death.

Concise Review: Mesenchymal Stem Cell Treatment of the Complications of Diabetes Mellitus

Mesenchymal stem cells (MSCs) are multipotent, self‐renewing cells that can be found in almost all postnatal organs and tissues. The main functional characteristics of MSCs are their immunomodulatory ability, capacity for self‐renewal, and differentiation into mesodermal tissues. The ability of MSCs to differentiate into several cell types, including muscle, brain, vascular, skin, cartilage, and bone cells, makes them attractive as therapeutic agents for a number of diseases including complications of diabetes mellitus. We review here the potential of MSCs as new therapeutic agents in the treatment of diabetic cardiomyopathy, diabetic nephropathy, diabetic polyneuropathy, diabetic retinopathy, and diabetic wounds. Also, in this review we discuss the current limitations for MSCs therapy in humans. STEM CELLS 2011;29:5–10

IL-33/ST2 axis in inflammation and immunopathology

Interleukin-33 (IL-33), a member of the IL-1 family of cytokines, binds to its plasma membrane receptor, heterodimeric complex consisted of membrane-bound ST2L and IL-1R accessory protein, inducing NFkB and MAPK activation. IL-33 exists as a nuclear precursor and may act as an alarmin, when it is released after cell damage or as negative regulator of NFκB gene transcription, when acts in an intracrine manner. ST2L is expressed on several immune cells: Th2 lymphocytes, NK, NKT and mast cells and on cells of myeloid lineage: monocytes, dendritic cells and granulocytes. IL-33/ST2 axis can promote both Th1 and Th2 immune responses depending on the type of activated cell and microenvironment and cytokine network in damaged tissue. We previously described and discuss here the important role of IL-33/ST2 axis in experimental models of type 1 diabetes, experimental autoimmune encephalomyelitis, fulminant hepatitis and breast cancer. We found that ST2 deletion enhance the development of T cell-mediated autoimmune disorders, EAE and diabetes mellitus type I. Disease development was accompanied by dominantly Th1/Th17 immune response but also higher IL-33 production, which suggest that IL-33 in receptor independent manner could promote the development of inflammatory autoreactive T cells. IL-33/ST2 axis has protective role in Con A hepatitis. ST2-deficient mice had more severe hepatitis with higher influx of inflammatory cells in liver and dominant Th1/Th17 systemic response. Pretreatment of mice with IL-33 prevented Con A-induced liver damage through prevention of apoptosis of hepatocytes and Th2 amplification. Deletion of IL-33/ST2 axis enhances cytotoxicity of NK cells, production of IFN-γ in these cells and systemic production of IFN-γ, IL-17 and TNF-α, which leads to attenuated tumor growth. IL-33 treatment of tumor-bearing mice suppresses activity of NK cells, dendritic cell maturation and enhances alternative activation of macrophages. In conclusion, we observed that IL-33 has attenuated anti-inflammatory effects in T cell-mediated responses and that both IL-33 and ST2 could be further explored as potential therapeutic targets in treatment of immune-mediated diseases.

Interleukin‐33/ST2 axis promotes breast cancer growth and metastases by facilitating intratumoral accumulation of immunosuppressive and innate lymphoid cells

The role of IL‐33/ST2 pathway in antitumor immunity is unclear. Using 4T1 breast cancer model we demonstrate time‐dependent increase of endogenous IL‐33 at both the mRNA and protein levels in primary tumors and metastatic lungs during cancer progression. Administration of IL‐33 accelerated tumor growth and development of lung and liver metastases, which was associated with increased intratumoral accumulation of CD11b+Gr‐1+ TGF‐β1+ myeloid‐derived suppressor cells (MDSCs) that expressed IL‐13α1R, IL‐13‐producing Lin−Sca‐1+ST2+ innate lymphoid cells (ILCs) and CD4+Foxp3+ST2+IL‐10+ Tregs compared to untreated mice. Higher incidence of monocytic vs. granulocytic MDSCs and plasmocytoid vs. conventional dendritic cells (DCs) was present in mammary tumors of IL‐33‐treated mice. Intratumoral NKp46+NKG2D+ and NKp46+FasL+ cells were markedly reduced after IL‐33 treatment, while phosphate‐buffered saline‐treated ST2‐deficient mice had increased frequencies of these tumoricidal natural killer (NK) cells compared to untreated wild‐type mice. IL‐33 promoted intratumoral cell proliferation and neovascularization, which was attenuated in the absence of ST2. Tumor‐bearing mice given IL‐33 had increased percentages of splenic MDSCs, Lin−Sca‐1+ ILCs, IL‐10‐expressing CD11c+ DCs and alternatively activated M2 macrophages and higher circulating levels of IL‐10 and IL‐13. A significantly reduced NK cell, but not CD8+ T‐cell cytotoxicity in IL‐33‐treated mice was observed and the mammary tumor progression was not affected when CD8+ T cells were in vivo depleted. We show a previously unrecognized role for IL‐33 in promoting breast cancer progression through increased intratumoral accumulation of immunosuppressive cells and by diminishing innate antitumor immunity. Therefore, IL‐33 may be considered as an important mediator in the regulation of breast cancer progression.

Interleukin-1 receptor antagonist (IL-1Ra) and IL-1Ra producing mesenchymal stem cells as modulators of diabetogenesis

The increase of pro-inflammatory cytokines and oxidative stress leads to β-cell damage and promotes β-cells apoptosis, in types I and II of diabetes mellitus. Therefore, blocking of pro-inflammatory cytokines should be an effective way for the treatment of diabetes mellitus. When IL-1 occupies its receptor, various pro-inflammatory events are initiated including the synthesis and releases of chemokines and these chemokines attract neutrophils, macrophages, and lymphocytes that cause tissue inflammation. IL-1Ra is a naturally occurring cytokine and is the inhibitor of IL-1. When IL-1Ra binds to the IL-1 receptor, binding of IL-1 is blocked by IL-1Ra and pro-inflammatory signal from IL-1 receptor is stopped. There are mounting evidences to suggest that anti-inflammatory IL-1Ra reduces the inflammatory effects of IL-1 and preserves cell function in both types of diabetes. Therefore, IL-1Ra maybe a new therapeutic agent for diabetes mellitus types I and II. Mesenchymal stem cells (MSCs) are self-renewable multipotent stromal cells that have immunomodulatory capacity. Recently, well characterized subpopulations of MSCs which express IL-1Ra have been described. IL-1Ra expressed by these MSCs effectively binds to IL-1 receptor and protects tissues from inflammation-induced injuries. It has been previously shown that bone marrow-derived MSC therapy could be considered for the treatment of diabetes mellitus type 1 and complications of diabetes mellitus. This review presents understanding of potential use of IL-1Ra and MSCs as modulators of diabetogenesis.

Protective role of IL-33/ST2 axis in Con A-induced hepatitis

BACKGROUND & AIMS We used Concanavalin A-induced liver injury to study the role of Interleukin 33 and its receptor ST2 in the induction of inflammatory pathology and hepatocellular damage. METHODS We tested susceptibility to Concanavalin A induced hepatitis in ST2 deficient and wild type BALB/c mice and analyzed the effects of single injection of Interleukin 33 as evaluated by liver enzyme test, quantitative histology, mononuclear cell infiltration, cytokine production, intracellular staining of immune cells, and markers of apoptosis in the liver. RESULTS ST2 deficient mice developed significantly more severe hepatitis and had significantly higher number of mononuclear cells in the liver, CD4+ and CD8+ T cells, NKp46+ and CD3+NKp46+ cells, and F4/80+ macrophages. The level of pro-inflammatory cytokines in the sera and number of TNF alpha, IFN gamma, and IL-17 producing cells was higher in ST2 deficient mice. In contrast, number of CD4+Foxp3+ cells was statistically higher in wild type mice. Additionally, treatment of wild type mice with single (1 μg) injection of Interleukin 33 led to attenuation of the liver injury and milder infiltration of mononuclear cells, increase in total number of liver CD4+Foxp3+ cells and IL-4 producing CD4+ T cells. Interleukin 33 also suppressed the activation of caspase 3, prevented the expression of BAX, and enhanced the expression of antiapoptotic Bcl-2 in the liver. CONCLUSIONS We concluded that Interleukin 33/ST2 axis downregulated Concanavalin A-induced liver injury and should be evaluated as potential target in fulminant hepatitis in humans.

Concise Review: Therapeutic Potential of Mesenchymal Stem Cells for the Treatment of Acute Liver Failure and Cirrhosis

Currently, the most effective therapy for acute liver failure and advanced cirrhosis is liver transplantation. However, this procedure has several limitations, including lack of donors, surgical complications, immunological suppression, and high medical costs. The alternative approaches that circumvent the use of a whole liver, such as stem cell transplantation, have been suggested as an effective alternate therapy for hepatic diseases. Mesenchymal stem cells (MSCs), also known as multipotent mesenchymal stromal cells, are self‐renewing cells that can be found in almost all postnatal organs and tissues, including liver. During the past decade, great progress has been made in the field of MSC‐dependent liver regeneration and immunomodulation. Because of their potential for differentiation into hepatocytes as well as their immunomodulatory characteristics, MSCs are considered as promising therapeutic agents for the therapy of acute liver failure and cirrhosis. In this concise review, we have summarized therapeutic potential of MSCs in the treatment of acute liver failure and cirrhosis, emphasizing their regenerative and immunomodulatory characteristics after engraftment in the liver. We have also presented several outstanding problems including conflicting data regarding MSCs engraftment in the liver and unwanted mesenchymal lineage differentiation in vivo which limits MSC therapy as a mainstream treatment approach for liver regeneration. It can be concluded that efficient and safe MSC‐based therapy for acute and chronic liver failure remains a challenging issue that requires more investigation and continuous cooperation between clinicians, researchers, and patients. Stem Cells 2014;32:2818–2823

Elevated serum level of type-2 cytokine and low IL-17 in first episode psychosis and schizophrenia in relapse

Schizophrenia is chronic and debilitating mental disorder. In broad spectrum of possible causes or contributing factors, immune system and cytokines were investigated in the onset and development of schizophrenia. The aim of our study was to analyze the serum concentrations of type-1 cytokines: TNF-α, IFN-γ, type-2 cytokines: IL-4, IL-10, type-17 cytokine: IL-17 and regulatory cytokines: TGF-β, IL-27, IL-6, in drug-naive patients with First Episode Psychosis - FEP (n = 88) and Schizophrenia in relapse - SC in relapse patients (n = 45), comparing to healthy controls (n = 36). Also, we attempted to determine potential correlation between cytokine levels and/or cytokine ratios with clinical parameters, such as severity of illness, positive, negative and general psychopathology. Our results showed decreased levels of IL-17 (p = 0.018), demonstrating that type-17 response is blunted in psychotic episode. Increased levels of IL-4 (p = 0.033) showed that type-2 response is overweight in psychotic episode. Also, levels of IL-4 in serum of SC in relapse patients were higher than controls (p < 0.0005) and patient with FEP (p = 0.003). This alteration was accompanied with increase in production of TGF-β in psychotic patients (p = 0.009) and also in FEP (p < 0.0005) and SC in relapse (p < 0.0005). Analysis showed that TGF-β can be a valuable marker for psychosis. The presence of enhanced anti-inflammatory/immunosuppressive activity in schizophrenia may be an attempt to counteract or limit ongoing pro-inflammatory processes and downregulating chronic inflammation. Finally we have documented decreased levels of IL-17 and IL-17/TGF-β ratio in these types of psychotic patients, suggesting the new aspects of schizophrenia pathophysiology.

Galectin-3 Deficiency Accelerates High-Fat Diet Induced Obesity and Amplifies Inflammation in Adipose Tissue and Pancreatic Islets

Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet–induced obesity and diabetes. Obese LGALS3−/− mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3−/− mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c+CD11b+ macrophages and decreased CD4+CD25+FoxP3+ regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1β (IL-1β) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3−/− animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3−/− peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1–dependent IL-1β production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3−/− macrophages with NLRP3 small interfering RNA attenuated IL-1β production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes.

The roles of Galectin-3 in autoimmunity and tumor progression

Galectin-3, a unique chimera-type member of the β-galactoside-binding soluble lectin family, is widely expressed in numerous cells. Here, we discuss the role of Galectin-3 in T-cell-mediated inflammatory (auto) immunity and tumor rejection by using Galectin-3-deficient mice and four disease models of human pathology: experimental autoimmune encephalomyelitis (EAE), Con-A-induced hepatitis, multiple low-dose streptozotocin-induced diabetes (MLD-STZ diabetes) and metastatic melanoma. We present evidence which suggest that Galectin-3 plays an important pro-inflammatory role in Con-A-induced hepatitis by promoting the activation of T lymphocytes, NKT cells and DCs, cytokine secretion, prevention of M2 macrophage polarization and apoptosis of mononuclear cells, and it leads to severe liver injury. In addition, experiments in Galectin-3-“knock-out” mice indicate that Galectin-3 is also involved in immune-mediated β-cell damage and is required for diabetogenesis in MLD-STZ model by promoting the expression of IFN-gamma, TNF-alpha, IL-17 and iNOS in immune and accessory effector cells. Next, our data demonstrated that Galectin-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-gamma synthesis, but decreasing IL-10 production. Finally, based on our findings, we postulated that expression of Galectin-3 in the host may also facilitate melanoma metastasis by affecting tumor cell adhesion and modulating anti-melanoma immune response, in particular innate antitumor immunity. Taken together, we discuss the evidence of pro-inflammatory and antitumor activities of Galectin-3 and suggest that Galectin-3 may be an important therapeutic target.