Nemanja Jovicic

Differential Immunometabolic Phenotype in Th1 and Th2 Dominant Mouse Strains in Response to High-Fat Feeding

Immune reactivity plays an important role in obesity-associated metabolic disorders. We investigated immunometabolic phenotype of C57Bl/6 and BALB/c mice, prototypical Th1 and Th2-type strains, fed chow or high-fat diet (HFD) for 24 weeks. In comparison to C57Bl/6 mice, chow-fed BALB/c mice had higher body weight and weight gain, lower glycemia, more pronounced liver steatosis, but less inflammation and collagen deposition in liver. In response to HFD C57Bl/6 mice exhibited higher weight gain, higher glycemia, HbA1c and liver glycogen content, increased amount of visceral adipose tissue (VAT) and number of VAT associated CD3+CXCR3+ T cells, CD11c+ dendritic cells (DCs) and F4/80+ macrophages than BALB/c mice. More numerous CD3+ and CD8+ T lymphocytes, myeloid DCs, proinflammatory macrophages (F4/80+CD11b+CD11+ and F4/80+IL-1β+) and CD11b+Ly6Chigh monocytes and higher levels of proinflammatory IL-6, TNF-α and IFN-γ were present in liver in HFD-fed C57Bl/6 mice compared with diet-matched BALB/c mice. As opposed to C57Bl/6 mice, HFD induced marked liver steatosis and upregulated the hepatic LXRα and PPARγ genes in BALB/c mice. C57Bl/6 mice fed HFD developed liver fibrosis and increased hepatic procollagen and TGF-β mRNA expression, and IL-33, IL-13 and TGF-β levels in liver homogenates, while BALB/c mice fed HFD had scarce collagen deposition in liver. The obtained results suggest inherent immunometabolic differences in C57Bl/6 and BALB/c mice. Moreover, HFD Th1-type mice on high fat diet regimen are more susceptible to adiposity, liver inflammation and fibrosis, while Th2-type mice to liver steatosis, which is associated with differential immune cell composition in metabolic tissues. Strain-dependent differences in immunometabolic phenotype may be relevant for studies of obesity-associated metabolic diseases in humans.

Ethical and Safety Issues of Stem Cell-Based Therapy

Results obtained from completed and on-going clinical studies indicate huge therapeutic potential of stem cell-based therapy in the treatment of degenerative, autoimmune and genetic disorders. However, clinical application of stem cells raises numerous ethical and safety concerns. In this review, we provide an overview of the most important ethical issues in stem cell therapy, as a contribution to the controversial debate about their clinical usage in regenerative and transplantation medicine. We describe ethical challenges regarding human embryonic stem cell (hESC) research, emphasizing that ethical dilemma involving the destruction of a human embryo is a major factor that may have limited the development of hESC-based clinical therapies. With previous derivation of induced pluripotent stem cells (iPSCs) this problem has been overcome, however current perspectives regarding clinical translation of iPSCs still remain. Unlimited differentiation potential of iPSCs which can be used in human reproductive cloning, as a risk for generation of genetically engineered human embryos and human-animal chimeras, is major ethical issue, while undesired differentiation and malignant transformation are major safety issues. Although clinical application of mesenchymal stem cells (MSCs) has shown beneficial effects in the therapy of autoimmune and chronic inflammatory diseases, the ability to promote tumor growth and metastasis and overestimated therapeutic potential of MSCs still provide concerns for the field of regenerative medicine. This review offers stem cell scientists, clinicians and patients useful information and could be used as a starting point for more in-depth analysis of ethical and safety issues related to clinical application of stem cells.

Galectin-3 Ablation Enhances Liver Steatosis, but Attenuates Inflammation and IL-33-Dependent Fibrosis in Obesogenic Mouse Model of Nonalcoholic Steatohepatitis.

The importance of Galectin-3 (Gal-3) in obesity-associated liver pathology is incompletely defined. To dissect the role of Gal-3 in fibrotic nonalcoholic steatohepatitis (NASH), Gal-3-deficient (LGALS3−/−) and wild-type (LGALS3+/+) C57Bl/6 mice were placed on an obesogenic high fat diet (HFD, 60% kcal fat) or standard chow diet for 12 and 24 wks. Compared to WT mice, HFD-fed LGALS3−/− mice developed, in addition to increased visceral adiposity and diabetes, marked liver steatosis, which was accompanied with higher expression of hepatic PPAR-γ, Cd36, Abca-1 and FAS. However, as opposed to LGALS3−/− mice, hepatocellular damage, inflammation and fibrosis were more extensive in WT mice which had an elevated number of mature myeloid dendritic cells, proinflammatory CD11b+Ly6Chi monocytes/macrophages in liver, peripheral blood and bone marrow, and increased hepatic CCL2, F4/80, CD11c, TLR4, CD14, NLRP3 inflammasome, IL-1β and NADPH-oxidase enzymes mRNA expression. Thus, obesity-driven greater steatosis was uncoupled with attenuated fibrotic NASH in Gal-3-deficient mice. HFD-fed WT mice had a higher number of hepatocytes that strongly expressed IL-33 and hepatic CD11b+IL-13+ cells, increased levels of IL-33 and IL-13 and up-regulated IL-33, ST2 and IL-13 mRNA in liver compared with LGALS3−/− mice. IL-33 failed to induce ST2 upregulation and IL-13 production by LGALS3−/− peritoneal macrophages in vitro. Administration of IL-33 in vivo enhanced liver fibrosis in HFD-fed mice in both genotypes, albeit to a significantly lower extent in LGALS3−/− mice, which was associated with less numerous hepatic IL-13-expressing CD11b+ cells. The present study provides evidence of a novel role for Gal-3 in regulating IL-33-dependent liver fibrosis.

Mesenchymal stem cell‐derived factors: Immuno‐modulatory effects and therapeutic potential

Stem cell‐based therapy is considered to be a new hope in transplantation medicine. Among stem cells, mesenchymal stem cells (MSCs) are, due to their differentiation and immuno‐modulatory characteristics, the most commonly used as therapeutic agents in the treatment of immune‐mediated diseases. MSCs migrate to the site of inflammation and modulate immune response. The capacity of MSC to alter phenotype and function of immune cells are largely due to the production of soluble factors which expression varies depending on the pathologic condition to which MSCs are exposed. Under inflammatory conditions, MSCs‐derived factors suppress both innate and adaptive immunity by attenuating maturation and capacity for antigen presentation of dendritic cells, by inducing polarization of macrophages towards alternative phenotype, by inhibiting activation and proliferation of T and B lymphocytes and by reducing cytotoxicity of NK and NKT cells. In this review, we emphasized current findings regarding immuno‐modulatory effects of MSC‐derived factors and emphasize their potential in the therapy of immune‐mediated diseases.

Galectin-3 and IL-33/ST2 axis roles and interplay in diet-induced steatohepatitis

Immune reactivity and chronic low-grade inflammation (metaflammation) play an important role in the pathogenesis of obesity-associated metabolic disorders, including type 2 diabetes and nonalcoholic fatty liver disease (NAFLD), a spectrum of diseases that include liver steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Increased adiposity and insulin resistance contribute to the progression from hepatic steatosis to NASH and fibrosis through the development of proinflammatory and profibrotic processes in the liver, including increased hepatic infiltration of innate and adaptive immune cells, altered balance of cytokines and chemokines, increased reactive oxygen species generation and hepatocellular death. Experimental models of dietary-induced NAFLD/NASH in mice on different genetic backgrounds or knockout mice with different immune reactivity are used for elucidating the pathogenesis of NASH and liver fibrosis. Galectin-3 (Gal-3), a unique chimera-type β-galactoside-binding protein of the galectin family has a regulatory role in immunometabolism and fibrogenesis. Mice deficient in Gal-3 develop pronounced adiposity, hyperglycemia and hepatic steatosis, as well as attenuated liver inflammation and fibrosis when fed an obesogenic high-fat diet. Interleukin (IL)-33, a member of the IL-1 cytokine family, mediates its effects through the ST receptor, which is present on immune and nonimmune cells and participates in immunometabolic and fibrotic disorders. Recent evidence, including our own data, suggests a protective role for the IL-33/IL-33R (ST2) signaling pathway in obesity, adipose tissue inflammation and atherosclerosis, but a profibrotic role in NASH development. The link between Gal-3 and soluble ST2 in myocardial fibrosis and heart failure progression has been demonstrated and we have recently shown that Gal-3 and the IL-33/ST2 pathway interact and both have a profibrotic role in diet-induced NASH. This review discusses the current evidence on the roles of Gal-3 and the IL-33/ST2 pathway and their interplay in obesity-associated hepatic inflammation and fibrogenesis that may be of interest in the development of therapeutic interventions to prevent and/or reverse obesity-associated hepatic inflammation and fibrosis.

Mesenchymal stem cells attenuate liver fibrosis by suppressing Th17 cells – an experimental study

This study investigates molecular and cellular mechanisms involved in mesenchymal stem cell (MSC)‐mediated modulation of IL‐17 signaling during liver fibrosis. Mice received CCl4 (1 μl/g intraperitoneally) twice/week for 1 month. MSCs (1 × 106), or MSC‐conditioned medium (MSC‐CM), were intravenously injected 24 h after CCl4 and on every 7th day. Liver fibrosis was determined by macroscopic examination, histological analysis, Sirius red staining, and RT‐PCR. Serum levels of cytokines, indoleamine 2,3‐dioxygenase (IDO), and kynurenine were determined by ELISA. Flow cytometry was performed to identify liver‐infiltrated cells. In vitro, CD4+ T cells were stimulated and cultured with MSCs. 1‐methyltryptophan was used for inhibition of IDO. MSCs significantly attenuated CCl4‐induced liver fibrosis by decreasing serum levels of inflammatory IL‐17, increasing immunosuppressive IL‐10, IDO, and kynurenine, reducing number of IL‐17 producing Th17 cells, and increasing percentage of CD4+IL‐10+ T cells. Injection of MSC‐CM resulted with attenuated fibrosis accompanied with the reduced number of Th17 cells in the liver and decreased serum levels of IL‐17. MSC‐CM promoted expansion of CD4+FoxP3+IL‐10+ T regulatory cells and suppressed proliferation of Th17 cells. This phenomenon was completely abrogated in the presence of IDO inhibitor. MSCs, in IDO‐dependent manner, suppress liver Th17 cells which lead to the attenuation of liver fibrosis.

Mesenchymal Stem Cells Promote Metastasis of Lung Cancer Cells by Downregulating Systemic Antitumor Immune Response

Since majority of systemically administered mesenchymal stem cells (MSCs) become entrapped within the lungs, we used metastatic model of lung cancer, induced by intravenous injection of Lewis lung cancer 1 (LLC1) cells, to investigate the molecular mechanisms involved in MSC-mediated modulation of metastasis. MSCs significantly augmented lung cancer metastasis, attenuate concentrations of proinflammatory cytokines (TNF-α, IL-17), and increase levels of immunosuppressive IL-10, nitric oxide, and kynurenine in sera of LLC1-treated mice. MSCs profoundly reduced infiltration of macrophages, TNF-α-producing dendritic cells (DCs), TNF-α-, and IL-17-producing CD4+ T cells but increased IL-10-producing CD4+ T lymphocytes in the lungs of tumor-bearing animals. The total number of lung-infiltrated, cytotoxic FasL, perforin-expressing, TNF-α-, and IL-17-producing CD8+ T lymphocytes, and NKG2D-expressing natural killer (NK) cells was significantly reduced in LLC1 + MSC-treated mice. Cytotoxicity of NK cells was suppressed by MSC-conditioned medium. This phenomenon was abrogated by the inhibitors of inducible nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO), suggesting the importance of iNOS and IDO for MSC-mediated suppression of antitumor cytotoxicity of NK cells. This study provides the evidence that MSCs promote lung cancer metastasis by suppressing antitumor immune response raising concerns regarding safety of MSC-based therapy in patients who have genetic susceptibility for malignant diseases.

Mesenchymal Stem Cells Attenuate Cisplatin-Induced Nephrotoxicity in iNOS-Dependent Manner

Mesenchymal stem cells (MSCs) are, due to their immunomodulatory characteristics, utilized in therapy of immune-mediated diseases. We used murine model of cisplatin nephrotoxicity to explore the effects of MSCs on immune cells involved in the pathogenesis of this disease. Intraperitoneal application of MSCs significantly attenuated cisplatin nephrotoxicity, decreased inflammatory cytokines TNF-α and IL-17, and increased anti-inflammatory IL-10, IL-6, nitric oxide (NO), and kynurenine in sera of cisplatin-treated mice. MSC treatment significantly attenuated influx of leukocytes, macrophages, dendritic cells (DCs), neutrophils, CD4+ T helper (Th), and CD8+ cytotoxic T lymphocytes (CTLs) in damaged kidneys and attenuated the capacity of renal-infiltrated DCs, CD4+ Th, and CD8+ CTLs to produce TNF-α and IL-17. Similar effects were observed after intraperitoneal injection of MSC-conditioned medium (MSC-CM) indicating that MSCs exert their beneficial effects in paracrine manner. Inhibition of inducible nitric oxide synthase (iNOS) in MSC-CM resulted with increased number of TNF-α-producing DCs and IL-17-producing CTLs, decreased number of IL-10-producing tolerogenic DCs and regulatory CD4+FoxP3+ T cells, and completely diminished renoprotective effects of MSC-CM. In conclusion, MSCs, in iNOS-dependent manner, attenuated inflammation in cisplatin nephrotoxicity by reducing the influx and capacity of immune cells, particularly DCs and T lymphocytes, to produce inflammatory cytokines.

ST2 deletion increases inflammatory bone destruction in experimentally induced periapical lesions in mice.

INTRODUCTION ST2 is a member of the interleukin (IL)-1 receptor family, and IL-33 is its natural ligand. ST2 signaling promotes Th2 immune response in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. The purpose of this study was to investigate whether ST2 gene deletion affects the development of experimentally induced periapical lesions in mice. METHODS Pulps of mandibular molars from wild-type (WT) and ST2 knockout (ST2(-)/(-)) BALB/c mice were exposed and left open to the oral environment. After death, hemi-mandibles were isolated and prepared for histologic, immunohistochemical, and flow cytometric analysis. RESULTS The expression of IL-33 and its receptor ST2 was higher in periapical lesions in WT mice compared with normal root apices (both P < .05). The increased periapical bone loss observed in ST2(-)/(-) mice was associated with enhanced influx of neutrophils, CD3+ CXCR3+ Th1 cells, and CD3+ CCR6+ Th17 cells and increased number of tartrate-resistant acid phosphatase+ osteoclasts (all P < .05). Furthermore, periapical lesions in ST2(-)/(-) mice contained increased percentages of T cells expressing interferon-γ, IL-17, tumor necrosis factor-α, and IL-6 (all P < .05). In comparison with WT mice, CD3+ receptor activator of nuclear factor kappa B ligand+ T cells were increased, whereas CD3+ osteoprotegerin+ T cells were decreased in the lesions of ST2(-)/(-) mice (both P < .05). CONCLUSIONS ST2 deletion increases inflammatory bone loss in experimental periapical lesions in mice, which is associated with enhanced Th1/Th17 cell mediated periapical immune responses and increased osteoclastogenesis.

Stem Cells Therapy for Spinal Cord Injury

Spinal cord injury (SCI), a serious public health issue, most likely occurs in previously healthy young adults. Current therapeutic strategies for SCI includes surgical decompression and pharmacotherapy, however, there is still no gold standard for the treatment of this devastating condition. Inefficiency and adverse effects of standard therapy indicate that novel therapeutic strategies are required. Because of their neuroregenerative and neuroprotective properties, stem cells are a promising tool for the treatment of SCI. Herein, we summarize and discuss the promising therapeutic potential of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and ependymal stem/progenitor cells (epSPC) for SCI.